Compounds And Methods For Treating Fibrotic Pathologies

ABSTRACT

The present application provides methods for treating or preventing diseases and conditions associated with tissue fibrosis.

CLAIM OF PRIORITY

This application claims priority under U.S. Provisional Patent Application Ser. No. 62/880,494, filed on Jul. 30, 2019, and U.S. Provisional Patent Application Ser. No. 62/880,594, filed on Jul. 30, 2019, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to compounds (e.g., isochromane compounds) that inhibit YAP/TAZ in fibroblasts and are useful in treating diseases and conditions associated with tissue fibrosis.

BACKGROUND

Tissue fibrosis across all organs affects a vast population of people. In the U.S. alone over half a million people are affected by liver (>400k) and lung (>100k) fibrosis. These diseases remain very challenging to treat clinically. In examples such as idiopathic pulmonary fibrosis (IPF) and scleroderma, therapeutic options are extremely limited. In fact, for this group of diseases, the five year survival rate can be as bleak as many late stage aggressive cancers.

SUMMARY

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, and ultimately leads to fatal, end-stage organ scarring. Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) play a role in the mesenchymal cell activation that drives tissue fibrosis (Refs. 1-4). YAP and TAZ are downstream transcriptional effectors of multiple pro-fibrotic stimuli in mesenchymal cells (See e.g., Ref. 5), and their expression in other cells and tissues is essential to regeneration and homeostasis (See e.g., Ref. 6), complicating efforts to target them therapeutically (See e.g., Ref. 7).

In one general aspect, the present application provides methods for inhibiting YAP and TAZ in mesenchymal cells via GPCR agonism. The data presented herein demonstrates the efficacy of this approach in murine models of lung and liver fibrosis. Gα_(s)-coupled dopamine receptor D1 is preferentially expressed in lung and liver mesenchymal cells relative to other major resident cells of these organs. Agonism of the D1 receptor selectively inhibits YAP/TAZ function in mesenchymal cells, and shifts their phenotype in a YAP/TAZ dependent fashion from pro-fibrotic to fibrosis-resolving, effectively reversing in vitro extracellular matrix accumulation and stiffening and reversing in vivo tissue fibrosis. This finding demonstrates a cell-selective approach to inhibiting a target that drives tissue fibrosis, and establishes Gα_(s) agonism as a strategy for generating a fibrosis-resolving mesenchymal phenotype.

In one general aspect, the present application provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹ and R³ are as described herein.

In another general aspect, the present disclosure provides a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, R⁴ and R⁵ are as described herein.

In another general aspect, the present disclosure provides a compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein X¹, R¹, R³, and R⁴ are as described herein.

In another general aspect, the present disclosure provides a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein X¹, X², R¹, R², R⁴ and R⁵ are as described herein.

In another general aspect, the present disclosure provides a pharmaceutical composition comprising a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another general aspect, the present application provides a method of agonizing a Gα_(S) protein coupled receptor in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of promoting YAP/TAZ phosphorylation in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of inhibiting YAP/TAZ function in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of inhibiting expression of a profibrotic gene in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of reducing nuclear localization of YAP/TAZ in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of inhibiting expressing of α-smooth muscle actin (αSMA) in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of inhibiting extra-cellular matrix production and deposition by a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In another general aspect, the present application provides a method of enhancing extra-cellular matrix degradation by a cell, the method comprising contacting the cell with an effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof.

In yet another general aspect, the present application provides a method of treating or preventing a fibrotic pathology, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of the Formulae described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the present application will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 contains a schematic representation of molecular mechanism by which transcription factor YAP/TAZ promotes tissue fibrosis. YAP/TAZ nuclear localization and activity is mediated by multiple profibrotic signaling pathways. Activation of the D1 dopamine receptor inhibits nuclear localization and activity of YAP/TAZ in fibroblasts.

FIG. 2 contains a scheme showing that Gα_(s)-coupled Dopamine Receptor D1 is selectively expressed in pulmonary fibroblasts. The scheme shows regulation of YAP/TAZ transcription co-factor activity by GPCR signaling. Receptors which couple to Gα_(s) elevate cAMP and induce phosphorylation of YAP/TAZ which blocks nuclear localization. Receptors which couple to Galphα_(i/q/12) promote nuclear localization and activity of YAP/TAZ, e.g., through Rho-kinase (ROCK).

FIG. 3 contains a line plot showing GPCR expression profiling of cultured human alveolar epithelial cells and normal human pulmonary fibroblasts. Dopamine Receptor D1 (DRD1) transcripts are highly expressed in fibroblasts and not detected in epithelial cells. Red points indicate GPCRs which selectively couple to Gα_(s). Orange diagonal line indicates 10-fold preferential expression, blue line 100-fold.

FIG. 4 contains a bar graph showing DRD1 expression in cultured non-IPF associated fibroblasts, IPF patient-derived fibroblasts, normal human alveolar epithelial cells (NHAEpC), and normal human microvasculature endothelial cells (NHMVEC), passage 6 or less. NHAEpC and NHMVEC, n=2. non-IPF FB and IPF FB, n=6.

FIG. 5 shows expression of DRD1 in freshly isolated mouse lung fibroblasts (FB), epithelial (EpC), endothelial cells (EC), and leukocytes (Leuk). Lung from a healthy COL1A1-GFP expressing mouse was enzymatically digested then sorted for markers of fibroblasts (GFP+, CD140a+), epithelial cells (CD326+), endothelial cells (CD31+), and leukocytes (CD45+) followed by RNA isolation to validate selective populations and expression of Drd1.

FIG. 6 shows that dopamine Receptor D1 agonism blocks YAP/TAZ nuclear localization. Selective agonists of dopamine receptor D1 with varying efficacy inhibit YAP/TAZ nuclear localization; this effect can be overcome by treatment with a DRD1 receptor antagonist (SCH 39166, 3 μM), (Dihydrexidine, 10 μM). IMR-90 cells treated 2 hours prior to fixation. N=4 (****p<0.0001 vs. 0.10% DMSO vehicle control). Scale bar represents 100 μm.

FIG. 7 shows that, consistent with DRD1 expression, D1 agonist (e.g., DHX) inhibits YAP/TAZ only in fibroblasts (IPF-FBs) but not in epithelial (NHEpCs) or endothelial (NHMVECs) cells. N=4 (****p<0.0001 vs. 0.1% DMSO vehicle control)

FIG. 8 shows that YAP/TAZ localization is induced through multiple ligands which stimulate receptors coupled to Galphα_(i/q/12), endothelin 1 (ET-1: 100 nM), lysophosphatidic acid (LPA: 10 μM), and serotonin (5-HT: 1 μM). In each case DHX treatment (10 μM) can reverse this effect on YAP/TAZ. IMR-90 cells plated densely onto plastic cell culture plates for 24 hours in media containing 0.1% FBS, treated for 2 hours prior to fixation. N=4 (****p<0.0001 vs. 0.1% DMSO vehicle control), (++++p<0.0001 vs. the respective stimulated agonist ET-1, LPA, or 5-HT). Scale bar represents 100 μm.

FIG. 9 contains a bar graph showing cAMP measured in IPF patient-derived fibroblasts treated for 20 minutes with DHX (10 μM)+/−SCH 39166 (3 μM). N=3 (**p<0.01 vs. 0.1% DMSO vehicle control).

FIG. 10 shows phosphorylation of YAP by Rho-kinase inhibitor Y27632 (20 μM), direct cAMP stimulator Forskolin (10 μM), or DHX (10 μM). IMR-90 cells cultured for 24 hours in media containing 0.1% FBS, treated 2 hours prior to fixation. N=3 (***p<0.001 vs. 0.1% DMSO vehicle control).

FIG. 11 shows that D1 agonist (e.g., DHX) reverses fibroblast matrix deposition, contraction and stiffening. DHX blocks profibrotic gene expression in IPF patient-derived fibroblasts. Genes which encode Connective tissue growth factor (CTGF), Collagen I (COL1A1), αSMA (ACTA2), and Fibronectin (FN1) are reduced by 24 hour treatment with DHX (10 μM), +/−SCH 39166 (3 μM). N=3 (****p<0.0001, ***p<0.001, *p<0.05 vs. 0.1% DMSO vehicle control)

FIG. 12 shows that D1 agonist (e.g., DHX) reverses TGFβ-induced αSMA+ stress fiber formation. IMR-90 cells pre-stimulated with 2 ng/mL TGFβ for 48 hours and then treated with DHX (10 μM)+2 ng/mL TGFβ for an additional 24 hours prior to fixation. Cells which are positive for αSMA were quantified by a blinded investigator and noted in the bottom right corner, a minimum of 300 cells in each experiment were quantified. N=3.

FIG. 13 shows that D1 agonist (e.g., DHX) reverses TGFβ-induced extracellular matrix accumulation. IPF patient-derived fibroblasts grown at confluence were pre-stimulated with 2 ng/mL TGFβ for 48 hours and then treated with DHX (10 μM)+2 ng/mL TGFβ for an additional 24 hours prior to fixation. Cell derived matrix is measured using antibodies for Collagen I and Fibronectin. N=3 (****p<0.0001, ***p<0.001, **p<0.01 vs. 0.1% DMSO vehicle control) FIG. 14 shows that D1 agonist (e.g., DHX) attenuates IPF patient-derived fibroblast contractility measured by traction force microscopy. Representative traction maps are shown from cells plated onto 6.4 kPa matrices treated with the indicated concentration of DHX. RMS tractions were determined in two independent experiments; box and whisker plots show min to max, quartile, and mean from one representative experiment (****p<0.0001, *p<0.05 vs. 0.1% DMSO vehicle control).

FIG. 15 shows that D1 agonist (e.g., DHX) reverses extracellular matrix stiffening. NIH-3T3 cells plated onto gelatin-coated tissue culture plates stimulated to deposit matrix with 2 ng/mL TGFβ and 20 μM ascorbic acid for 72 hours prior to AFM microindentation analysis to measure stiffness (elastic modulus). The same dishes were then treated +/−10 μM DHX in the same media for another 72 hours and AFM analysis. The matrices were decellularized and passage 3 NHLFs were plated onto the matrices; after 24 hours RNA was collected and expression of profibrotic genes was analyzed. AFM analysis N=2. 75 indentation measurements were made for each plate. The box and whisker plots show min to max, quartile, and mean from one representative experiment (****p<0.0001 vs. 0.1% DMSO vehicle control) (++p<0.01, +p<0.05 vs. the same culture plate after the first 72 hour incubation). Measurement of RNA from cell plated onto decellularized matrices N=3. (*p<0.05 vs. 0.1% DMSO vehicle control).

FIG. 16 shows that D1 agonist (e.g., DHX) modulates matrix deposition vs. matrix degradation gene program. IPF patient-derived fibroblasts cultured in media containing 0.1% FBS were stimulated for 24 hours with 2 ng/mL TGFβ and 10 μM DHX prior to RNA isolation. Genes which encode for ECM crosslinking: transglutaminase 2 (TGM2), lysyl oxidase and lysyl oxidase-like enzymes (LOX and LOXL1-4), ECM degradation: uPA (PLA U), tPa (PLAT), cathepsin K (CTSK), and matrix metalloprotease-14 (MMP14) and ECM protease inhibitors: metalloprotease inhibitor 3 (TIMP3), and plasminogen activator inhibitor 1 (SERPINE1) were measured. N=3. (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. 0.1% DMSO vehicle control non-TGFβ treated), (++++p<0.0001, ++p<0.01, +p<0.05 vs. 0.10% DMSO vehicle control TGFβ treated).

FIG. 17 shows that D1 agonist (e.g., DHX) therapeutically reverses bleomycin-induced pulmonary fibrosis. Weight change as a result of bleomycin induced lung injury and DHX therapeutic benefit. Female mice were intratracheally administered Bleomycin on Day 0; treatment was initiated on Day 10 (5 mg/kg DHX i.n., daily) and continued until day 24.

FIG. 18 shows H&E staining to visualize collagen and architectural changes. Paraffin embedded lung sections were stained and analyzed in a blinded fashion by a pulmonary pathologist and scored using the Ashcroft method.

FIG. 19 shows results of hydroxyproline assay to measure collagen deposition in the lungs. Snap frozen lung tissue was biochemically analyzed for collagen abundance using the hydroxyproline assay.

FIG. 20 contains immunofluorescence imaging of lung sections for αSMA and Yap/Taz. Lung sections were stained immune-probed for αSMA and Yap/Taz. Cells which were double positive for both αSMA and Yap/Taz were quantified using automated software.

FIG. 21 shows changes in pro-fibrotic gene expression, Yap and Taz (Wwtr1) in whole lung homogenates. Sham Control N=15, Bleo Control N=17, and Bleo DHX N=17. (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. Sham Control) (++p<0.01, +p<0.05 vs. the respective Bleo Control).

FIG. 22 shows that intratracheally administered YAP/TAZ siRNA worsens bleomycin induced lung injury and fibrosis. Mice were administered bleomycin on Day 0 and then intratracheally administered siRNA for Yap and Taz on Day 14. On Day 21 BAL fluid was collected and lungs were harvested for analysis. Yap/Taz siRNA enhanced collagen deposition. Sham treated mice N=4, Bleo treated mice N=6 (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. Sham NT-siRNA).

FIG. 23A contains images of lungs treated with bleomycin and YAP/TAZ siRNA. FIG. 23B contains a bar graph showing weight of sham- and bleo-treated lungs. FIG. 23C contains a bar graph showing total BAL fluid protein in sham- and bleo-treated lungs.

FIG. 24 shows that D1 agonist (e.g., DHX) inhibits YAP/TAZ localization in fibroblasts from multiple tissues. Mesenchymal cells derived from lung (IMR-90 and IPF-FBs) hepatic stellate cells (HSC), human adult cardiac fibroblasts, (HACF) and human dermal fibroblasts (HDF) were plated densely (Confluent) or sparsely (Control and all remaining conditions) onto plastic cell culture plates for 24 hours in media containing 0.10% FBS, treated for 2 hours with Rho kinase inhibitor Y27632, adenylate cyclase activator forskolin and DHX. N=4, Cells that were positive for nuclear YAP/TAZ were quantified by automated image analysis.

FIG. 25 shows time course for D1 agonist (e.g., DHX) inhibition of YAP/TAZ nuclear localization. IMR-90 cells, N=3 (***p<0.001 vs. 0.1% DMSO vehicle control).

FIG. 26 shows that D1 agonist (e.g., DHX) does not lose potency in IPF derived fibroblasts, unlike PGE2. N=3.

FIG. 27 shows that D1 agonist (e.g., DHX) inhibits pro-fibrotic gene expression through DRD1 agonism. siRNA treatment to knockdown DRD1.

FIG. 28 shows reduced D1 agonist (e.g., DHX)-mediated inhibition of YAP/TAZ nuclear localization and pro-fibrotic gene expression in DRD1-siRNA treated cells. IMR-90 cells transfected with siRNA targeting DRD1 or NT siRNA for 72 hours prior to 2 hour (b) or 24 hour (c) treatment with DHX. N=2 for all (****p<0.0001, **p<0.01, *p<0.05 vs. NTsiRNA).

FIG. 29 shows that D1 agonist (e.g., DHX) inhibition of pro-fibrotic gene expression and matrix deposition requires inhibition of YAP/TAZ. DHX does not inhibit pro-fibrotic gene expression when fibroblasts express a constitutively active mutant TAZ (TAZ4SA). TAZ4SA expression was induced with 100 ng/mL doxycycline for 72 hours prior to treatment with 10 μM or the indicated concentration of dihydrexidine. For gene expression experiments efficacy of DHX in NIH-3T3 cells was validated with by DHX (10 μM) effect on TGFβ (24 hour, 2 ng/mL) induced gene expression. N=2 for all.

FIG. 30 shows that D1 agonist (e.g., DHX) inhibition of pro-fibrotic gene expression and matrix deposition requires inhibition of YAP/TAZ. DHX does not inhibit ECM deposition when fibroblasts express a constitutively active mutant TAZ (TAZ4SA). TAZ4SA expression was induced with 100 ng/mL doxycycline for 72 hours prior to treatment with 10 μM or the indicated concentration of dihydrexidine. For gene expression experiments efficacy of DHX in NIH-3T3 cells was validated with by DHX (10 μM) effect on TGFβ (24 hour, 2 ng/mL) induced gene expression. N=2 for all.

FIG. 31 shows that D1 agonist (e.g., DHX) alone does not affect lung matrix content or profibrotic gene expression. In normal healthy mice DHX did not alter body weight. n=6 mice per group.

FIG. 32 shows that in normal healthy mice D1 agonist (e.g., DHX) did not alter lung histology n=6 mice per group.

FIG. 33 shows that in normal healthy mice D1 agonist (e.g., DHX) did not alter lung collagen deposition. n=6 mice per group.

FIG. 34 shows that in normal healthy mice D1 agonist (e.g., DHX) did not alter expression of Ctgf, Col1a1, Acta2, and Fn1. n=6 mice per group.

FIG. 35 shows that D1 agonist (e.g., DHX) reverses hepatic stellate cell activation and in vivo hepatic fibrosis. DRD1 is preferentially expressed in cultured human hepatic stellate cells (HSCs): ACTA2, PDGFRA positive, relative to cultured human hepatocytes (Heps): ALB positive. N=1.

FIG. 36 shows that D1 agonist (e.g., DHX) inhibits TGFβ-mediated hepatic stellate cell activation in vitro. HSCs were stimulated with TGFβ for 48 hours+/−DHX (10 μM) prior to total protein isolation and western blot analysis of αSMA and Fibronectin. N=3 (***p<0.001, **p<0.01, *p<0.05 vs. Control+TGFβ).

FIG. 37 shows that D1 agonist (e.g., DHX) reverses bile duct ligation (BDL)-mediated fibrosis in vivo measured by trichrome staining. Sham Control N=5, BDL Control N=7, and BDL DHX N=8 (**p<0.01 vs. BDL Control).

FIG. 38 shows that D1 agonist (e.g., DHX) reverses Bile duct ligation (BDL) mediated fibrosis in vivo measured by hydroxyproline. Sham Control N=5, BDL Control N=7, and BDL DHX N=8 (**p<0.01 vs. BDL Control).

FIG. 39 shows that YAP phosphorylation blocks nuclear localization of pYAP.

FIG. 40 shows that D1 receptor agonists targeting the same receptor and having diverse structures produce similar effect.

FIG. 41 shows that YAP inactivation by D1 agonist (e.g., DHX) are based on D1 receptor activity.

FIG. 42 shows that D1 receptor agonist reduces expression of multiple profibrotic genes in fibroblasts from patients with IPF.

FIG. 43 shows that D1 receptor agonist (e.g., DHX) selectively slows proliferation in IPF fibroblasts. IPF fibroblast and Normal lung fibroblast co-culture proliferation. Cell are prelabelled with fluorescent dyes (red and green) and then cocultured at a ratio of about 1:1 in 96 well plates. Cell counts are determined every 24 hours and plotted as a ratio of IPF/HLF cells. In the control wells the IPF cells outgrow and take over the well. N=2.

FIG. 44 shows that YAP/TAZ are necessary for matrix stiffness-dependent fibroblast activation.

FIG. 45 shows that mutant YAP/TAZ are active on soft matrices in NIH 3T3 fibroblasts.

FIG. 46 shows that YAP/TAZ confer fibrogenic potential in vivo.

FIG. 47 shows that global YAP/TAZ targeting is not viable.

FIG. 48 shows Western blot protein expression of the D1 dopamine receptor from IPF patient derived fibroblasts, normal human alveolar epithelial cells (NHAEpC), and normal human microvasculature endothelial cells. NHAEpC and NHMVEC, N=2. non-IPF FB and IPF FB, N=3 different donor lines.

FIG. 49 shows GPCR expression profiling of primary cultured human pulmonary microvascular endothelial cells and normal human pulmonary fibroblasts. Red points indicate GPCRs that selectively couple to Gαs. Blue lines indicate 100-fold preferential expression. Prostaglandin receptors PTGER2 and PTGDR (red points directly above DRD1) were also selectively expressed in fibroblasts vs. endothelial cells, however both of these receptors were highly expressed in epithelial cells.

FIG. 50A shows that dopamine receptor D1 agonism blocks YAP/TAZ nuclear localization. D1 receptor selective agonists inhibit YAP/TAZ nuclear localization. IPF patient-derived lung fibroblasts cells treated 2 hours prior to fixation with diverse dopaminergic agonists (10 μM). N=4 different patient samples. % nuclear localization of YAP/TAZ was determined using automated imaging software. Scale bar represents 100 μm.

FIG. 50B shows cAMP measured in IPF patient-derived fibroblasts treated for 20 minutes with D1 receptor agonist (e.g., DHX). N=3.

FIG. 50C shows that D1 receptor agonist (e.g., DHX) inhibits YAP/TAZ nuclear localization in fibroblasts from multiple organs: hepatic stellate cells (HSC), human adult cardiac fibroblasts (HACFs), and human dermal fibroblasts (HDFs) but not in lung alveolar epithelial (NHAEp) or endothelial (NHMVE) cells. N=3 (****p<0.0001 vs. 0.1% DMSO vehicle control).

FIG. 51 shows that D1 receptor agonist (e.g., DHX) reverses fibroblast matrix deposition, contraction and stiffening. D1 receptor selective agonists inhibit fibroblast activation (Representative image: 1 μM dihydrexidine (DHX). IPF patient-derived lung fibroblasts cells treated for 72 hours prior to fixation with a library of diverse, mixed selectivity dopaminergic agonists (1 μM)+TGFβ. N=4 different patient samples. αSMA intensity was determined using automated imaging software. Scale bar represents 100 μm.

FIG. 52 shows that D1 receptor agonist (e.g., DHX) attenuates IPF fibroblast contractility measured by traction force microscopy. (****p<0.0001, *p<0.05 vs. 0.1% DMSO vehicle control).

FIG. 53 shows that D1 receptor agonist (e.g., DHX) reverses extracellular matrix accumulation. IPF patient-derived fibroblasts pre-stimulated with 2 ng/mL TGFβ for 48 hours, then treated with DHX+2 ng/mL TGFβ for additional 24 hours. N=3 (****p<0.0001, ***p<0.001, **p<0.01 vs. 0.1% DMSO vehicle control)

FIG. 54 shows that D1 receptor agonist (e.g., DHX) and YAP/TAZ siRNA modulate matrix crosslinking and degradation gene programs. IPF fibroblasts treated 24 hours with 2 ng/mL TGFβ+/−10 μM DHX or YAP and TAZ siRNA (>90% knockdown). N=3. Heat map indicates % change relative to unstimulated controls.

FIG. 55 shows that D1 receptor agonist (e.g., DHX) reverses extracellular matrix stiffening. IPF patient derived fibroblasts and their cell-derived matrices were characterized by AFM microindentation using a spherical tip after 72 hours, then treated+/−10 μM DHX in matrix deposition media for additional 72 hours and re-characterized. N=5 different patient samples. (*p<0.05 vs. 0.10% DMSO vehicle control).

FIG. 56 shows that DHX selectively blocks expression of YAP/TAZ target genes in lung fibroblasts in vivo. Two groups of mice were injured intratracheally with bleomycin at day 0, on day 10 one group received two doses of DHX (2 and 24 hours prior to collecting lungs) and the other received vehicle control. On day 11 lungs were collected to flow sort fibroblasts, epithelial, and endothelial cells.

FIG. 57 shows changes in RNA expression of YAP/TAZ target genes from freshly isolated cells.

FIG. 58A shows that DRD1 agonism selectively blocks localization and activity of YAP/TAZ in lung fibroblasts. IPF derived lung fibroblasts, lung alveolar epithelial (NHAEp) and endothelial (NHMVE) cells were treated for 2 hours with DRD1 selective agonist (e.g., DHX)

FIG. 58B shows results of an experiment where IPF derived lung fibroblasts, lung alveolar epithelial (NHAEp) and endothelial (NHMVE) cells were treated for 24 hours with DRD1 selective agonist prior to RNA isolation and measurement of YAP/TAZ target genes. N=2 technical replicates.

FIG. 58C shows results of an experiment where IPF derived lung fibroblasts, lung alveolar epithelial (NHAEp) and endothelial (NHMVE) cells were treated for 2 hours with butaprost (EP2 receptor agonist). DRD1 receptor is only expressed in fibroblasts while EP2 receptor, which also elevates cAMP, is expressed in all three cell types.

FIG. 58D shows results of an experiment where IPF derived lung fibroblasts, lung alveolar epithelial (NHAEp) and endothelial (NHMVE) cells were treated for 24 hours with butaprost (EP2 receptor agonist) prior to RNA isolation and measurement of YAP/TAZ target genes. N=2 technical replicates.

FIG. 59A shows that D1 receptor agonist (e.g., DHX) activity is dependent on DRD1 receptor.

FIG. 59B shows that D1 receptor agonist (e.g., DHX) activity is dependent on DRD1 receptor.

FIG. 59C shows that DRD1 siRNA treatment blocks DHX's elevation of cAMP. N=3, IMR-90 lung fibroblasts.

FIG. 59D shows that DRD1 siRNA treatment blocks inhibition of YAP/TAZ nuclear localization. N=3, IMR-90 lung fibroblasts.

FIG. 59E shows that DRD1 siRNA treatment inhibition of YAP/TAZ target genes. N=3, IMR-90 lung fibroblasts.

FIG. 59F shows that dopamine receptor D1 antagonists SCH-39166 and LE-300 block DHX's elevation of cAMP. N=3, IMR-90 lung fibroblasts.

FIG. 59G shows that dopamine receptor D1 antagonists SCH-39166 and LE-300 block DHX's inhibition of YAP/TAZ nuclear localization. N=3, IMR-90 lung fibroblasts.

FIG. 59H shows that dopamine receptor D1 antagonists SCH-39166 and LE-300 block DHX's inhibition of YAP/TAZ target genes. N=3, IMR-90 lung fibroblasts.

FIG. 60A shows that DHX blocks proliferation and primes lung fibroblasts for apoptosis. Proliferation of lung fibroblasts (IMR-90 cells) measured for 4 days in the presence of GPCR agonists ET-1 (100 nM) and LPA (10 μM)+/−10 μM DHX. Cells were fixed and counted with DAPI at the end of each day. N=3 technical triplicates.

FIG. 60B shows results of an experiment where proliferation of lung fibroblasts (IPF Patient-Derived Lung fibroblasts) measured for 4 days in the presence of GPCR agonists ET-1 (100 nM) and LPA (10 μM)+/−10 μM DHX. Cells were fixed and counted with DAPI at the end of each day. N=3 technical triplicates.

FIG. 60C shows results of an experiment where proliferation of lung fibroblasts (IMR-90 cells) was measured for 4 days in the presence of growth factor TGFβ (2 ng/mL)+/−10 μM DHX. Cells were fixed and counted with DAPI at the end of each day. N=3 technical triplicates.

FIG. 60D shows results of an experiment where proliferation of lung fibroblasts (IPF Patient-Derived Lung fibroblasts) measured for 4 days in the presence of growth factor CTGF (100 ng/mL)+/−10 μM DHX. Cells were fixed and counted with DAPI at the end of each day. N=3 technical triplicates.

FIG. 60E shows results of an experiment where RNA Expression of pro-apoptotic factor BIM was assessed following 24 treatment with DHX (10 μM). N=4. IMR-90 cells.

FIG. 60F shows results of an experiment where RNA Expression of pro-apoptotic factor BIM was assessed following 24 treatment with DHX (10 μM). N=4. IPF Patient-Derived Lung fibroblasts.

FIG. 60G shows results of an experiment where RNA Expression of anti-apoptotic factor BCL2 was assessed following 24 treatment with DHX (10 μM). N=4. IMR-90 cells.

FIG. 60H shows results of an experiment where RNA Expression of anti-apoptotic factor BCL2 was assessed following 24 treatment with DHX (10 μM). N=4. IPF Patient-Derived Lung fibroblasts.

FIG. 61A DOPA decarboxylase is decreased in IPF, and correlates with worsening disease severity Expression levels for DDC and DRD1 were queried from microarray analyses of IPF (n=134) and control (n=108) lungs. Each data point represents expression levels from an individual. Bars indicate mean and standard deviation.

FIG. 61B shows results of an experiment where western blotting was used to detect DDC protein expression in whole lung homogenates from IPF (n=10) and control (n=11) lungs. Bars indicate mean and standard deviation.

FIG. 61C shows results of an experiment where univariate analysis of the correlation of DDC expression with forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) was performed using the Pearson's correlation coefficient (r). Each data point represents expression levels and lung function (expressed a percent predicted based on age, sex and ideal body weight) from an individual. P values are as indicated for each figure panel.

FIG. 62A shows that dopamine promotes anti-fibrotic effects. Dopamine inhibits YAP/TAZ nuclear localization in low density IPF-patient derived fibroblasts plated onto tissue culture plastic. N=2.

FIG. 62B shows that dopamine attenuates IPF fibroblast contractility measured by traction force microscopy (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. the indicated group).

FIG. 62C shows that dopamine reverses αSMA+ stress fiber formation. IPF-patient derived fibroblasts, pre-stimulated with 2 ng/mL TGFβ for 48 hours, then treated with dopamine (1 μM)+2 ng/mL TGFβ for additional 24 hours. N=4. Scale bar represents 500 μm.

FIG. 63 shows that DRD1 agonism blocks profibrotic gene expression in human dermal fibroblasts. Human dermal fibroblasts treated for 24 hours+/−2 ng/mL TGFβ and D1 agonists: DHX and A68930 prior to RNA isolation. N=2.

FIG. 64 shows that dihydrexidine (DHX) efficacy depends on expression of D1 like dopamine receptors (DRD1, DRD5). IMR-90 lung fibroblasts express higher levels of DRD1 and DRD5 than mesenchymal cells derived from uterine fibroids which results in marginal inhibition of YAP/TAZ nuclear localization by DHX in these cells. ND refers to the gene not being detected.

FIG. 65 contains a bar graph showing nuclear YAP/TAZ/DAPI inhibition by D1 agonists dihydrexidine (DHX), A-68930, (R)-(−)-apomorphine, and R(−)-2,10,11-trihydroxyaporphine. (10 μM) N=4 IPF-patient derived lung fibroblasts.

FIG. 66 contains a line plot showing that compound CTC-3 inhibits YAP/TAZ nuclear localization. Adult lung fibroblasts (N=2) sparsely plated into 96-well plates. Treated for 2 hours with compounds prior to fixing and immunostaining for YAP/TAZ. Imaging and quantification of nuclear YAP/TAZ performed through automation using a Cytation 5 (IC₅₀ is 102 nM).

FIG. 67 contains a line plot showing that compound CTC-6 inhibits YAP/TAZ nuclear localization. Adult lung fibroblasts (N=2) sparsely plated into 96-well plates. Treated for 2 hours with compounds prior to fixing and immunostaining for YAP/TAZ. Imaging and quantification of nuclear YAP/TAZ performed through automation using a Cytation 5 (IC₅₀ is 62 nM).

FIG. 68 contains a line plot showing that compound CTC-3 inhibits fibroblast proliferation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ and compounds at the indicated concentration. Proliferation determined by fixing and counting DAPI nuclei using a Cytation 5. N=2.

FIG. 69 contains a line plot showing that compound CTC-6 inhibits fibroblast proliferation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ and compounds at the indicated concentration. Proliferation determined by fixing and counting DAPI nuclei using a Cytation 5. N=2.

FIG. 70 contains a line plot showing that compound CTC-3 inhibits fibroblast activation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of αSMA intensity performed through automation using a Cytation 5. (IC₅₀ is 0.3 μM).

FIG. 71 contains a line plot showing that compound CTC-6 inhibits fibroblast activation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of αSMA intensity performed through automation using a Cytation 5. (IC₅₀ is 0.1 μM).

FIG. 72 contains a line plot showing that compound CTC-3 inhibits Collagen I deposition. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of Collagen I intensity performed through automation using a LI-COR Odyssey. (IC₅₀ is 0.7 μM).

FIG. 73 contains a line plot showing that compound CTC-6 inhibits Collagen I deposition. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of Collagen I intensity performed through automation using a LI-COR Odyssey. (IC₅₀ is 0.4 μM).

FIG. 74 contains a line plot showing that compound CTC-1 inhibits YAP/TAZ nuclear localization. Adult lung fibroblasts (N=2) sparsely plated into 96-well plates. Treated for 2 hours with compounds prior to fixing and immunostaining for YAP/TAZ. Imaging and quantification of nuclear YAP/TAZ performed through automation using a Cytation 5 (IC₅₀ is 285 nM).

FIG. 75 contains a line plot showing that compound CTC-2 inhibits YAP/TAZ nuclear localization. Adult lung fibroblasts (N=2) sparsely plated into 96-well plates. Treated for 2 hours with compounds prior to fixing and immunostaining for YAP/TAZ. Imaging and quantification of nuclear YAP/TAZ performed through automation using a Cytation 5 (IC₅₀ is 490 nM).

FIG. 76 contains bar graphs showing inhibition of profibrotic gene expression by compounds CTC-1 and CTC-2. IMR-90 lung fibroblasts, treated for 24 hours with the indicated concentration of compound. N=3.

FIG. 77A contains structure and YAP/TAZ inhibition efficacy of dihydrexidine (“DHX”). Human lung fibroblasts were treated for 2 hours with the indicated concentration of dihydrexidine prior to imaging and quantifying YAP/TAZ localization.

FIG. 77B contains an image of fried eggs model of predicted BBB penetration for DHX using TPSA and WLOGP properties. Compounds which fit into the “yolk” are predicted to enter the CNS. These predictions correlate with in vitro models of BBB penetration.

FIG. 78 contains structures and efficacy data for phenyl containing analogs of A-68930. Potency and Efficacy of 3-substituted A-68930 analogs. Measurements were obtained using gold-fish retina or rat striatum, per U.S. Pat. No. 5,621,133.

FIG. 79 contains comparative YAP/TAZ nuclear localization inhibition between A-68930 and D1 agonists of the present disclosure. Adult lung fibroblasts (N=2) sparsely plated into 96-well plates. Treated for 2 hours with compounds prior to fixing and immunostaining for YAP/TAZ. Imaging and quantification of nuclear YAP/TAZ performed through automation using a Cytation 5. A-68390 was tested as an optically pure stereoisomer. CTC-3 and CTC-6 are both 1:1 mixtures of active and inactive stereoisomers (the observed potency is 2-fold higher). The major drawback of A-68930 is it lacks full efficacy to elevate cAMP.

FIG. 80 contains line plots efficacy of CTC-6 in vitro. A. Dose-response curve for CTC-6 inhibiting YAP/TAZ localization in lung fibroblasts. B. Human lung fibroblasts were stimulated for 4 days with 2 ng/mL TGFβ and cell number was measured daily by counting DAPI nuclei from fixed cells treated with the indicated concentration of CTC-6. C. Expression of αSMA intensity measured by immunocytochemistry D. Collagen deposition measured using a “in-cell Western blot” technique developed by our group. Important to note: these data were collected using a racemic mixture of CTC-6. The active stereoisomer is 2-fold more potent that the inactive stereoisomer.

FIG. 81A contains fried eggs model of predicted BBB penetration using TPSA and WLOGP properties. Compounds which fit into the “yolk” are predicted to enter the CNS. DHX and CTC-6 are plotted along with the other compounds of the present disclosure.

FIG. 81B contains fried eggs model of predicted BBB penetration using TPSA and WLOGP properties. Compounds which fit into the “yolk” are predicted to enter the CNS. A-68930 and CTC-6 are plotted along with the other compounds of the present disclosure.

FIG. 82 contains compound structures and data from cyclohexane and cyclooctane derivatives of DHX. Potency appears to be maintained and the efficacy is dramatically increased in the cycloctane. Measurements were obtained using gold-fish retina or rat striatum according to published patent and manuscript.

FIG. 83 contains a fried eggs model of predicted BBB penetration using TPSA and WLOGP properties for the compounds of example 9 having various heterocyclis in position R¹ of Formula (I).

FIG. 84 shows compounds having dinapsoline/A-68930 hybrid scaffold (3), which is more potent than DHX and more efficacious than A-68930. Dinapsoline/A-68930 hybrid scaffolds were previously described by a group at Purdue University in 2010. This hybrid is ˜6× more potent than DHX and shows full efficacy at elevating cAMP.

FIG. 85 shows fried eggs model of predicted BBB penetration using TPSA and WLOGP properties of compound of Example 2. Chemical structure of the compound is also shown.

FIG. 86 shows that dinapsoline and dinoxyline display similar D1 binding affinity as dihydrexidine. Not shown in the figure, all three compounds produced full magnitude cAMP response (full efficacy) in published manuscripts. Dinapsoline (DNS) and dinoxyline (DNX) are dihydrexidine (DHX) framework mimics discovered in the late 1990s and early 2000s by the same group from Purdue University.

FIG. 87 shows fried eggs model of predicted BBB penetration using TPSA and WLOGP properties of compound of Example 1 and dinapsoline (DNS), dinoxyline (DNX), and dihydrexidine (DHX). Chemical structure of the compound of example 1 is also shown.

FIG. 88 contains line plots showing % nuclear YAP/TAZ for compound CTC-3 in human lung fibroblasts and human alveolar epithelial cells.

FIG. 89 contains line plots showing % nuclear YAP/TAZ for compound CTC-6 in human lung fibroblasts and human alveolar epithelial cells.

FIG. 90 contains chemical structure of compound 1 and also contains a line plot showing that compound 1 inhibits fibroblast proliferation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ and compounds at the indicated concentration. Proliferation determined by fixing and counting DAPI nuclei using a Cytation 5. N=2.

FIG. 91A contains a line plot showing that compound 1 inhibits fibroblast activation. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of αSMA intensity performed through automation using a Cytation 5.

FIG. 91B contains a line plot showing that compound 1 inhibits Collagen I deposition. Adult lung fibroblasts plated into 96-well plates, stimulated with 2 ng/mL TGFβ for 96 hours and treated with the indicated concentration of compounds every 48 hours. Imaging and quantification of Collagen I intensity performed through automation using a LI-COR Odyssey.

FIG. 92A shows in vitro efficacy of compound 1 (MS-9). Human lung fibroblasts and alveolar epithelial cells are treated with MS-9 and influence on YAP/TAZ localization is determined.

FIG. 92B shows in vitro efficacy of compound 1 (MS-9) blocks expression of TGFβ.

FIG. 92C contains a line plot showing cell count (filed of view) for 1 μM and 10 μM of compound 1 (MS-9).

DETAILED DESCRIPTION

Tissue fibrosis can occur in multiple vital organs including heart, lung, liver, and kidney. Fibrosis is a progressive process which, through multiple mechanisms, transforms a normal healthy organ into an architecturally and functionally compromised tissue. From a clinical standpoint they represent a serious problem as the therapeutic options remain minimal and the prognosis is generally very poor. Dopamine receptors, which are almost exclusively researched as part of the central nervous system, are actually highly expressed in the periphery as well in select tissues and cells in the body. These receptors signal through downstream pathways which play a major role in tissue fibrosis. One example of such receptors is a Gα_(s)-coupled receptor, such as a dopamine receptor D1 (DRD1). As described in the present disclosure, agonising dopamine receptors leads to treatment or prevention of tissue fibrosis in multiple organs.

YAP and TAZ are transcriptional co-activators and central effectors of the Hippo pathway (Ref. 8). Originally identified based on their roles in organ growth and size control during tissue morphogenesis, the Hippo pathway and YAP/TAZ in adult tissues regulate epithelial and endothelial homeostasis (Ref. 9-13), stem cell function (Ref. 14-16) and tissue regeneration (Ref. 9,17,18). Roles for YAP and TAZ in mesenchymal cell activation and fibrosis in multiple organs (Ref. 19-22), including the lung and liver (Ref. 2), was also shown. An array of mechanical and biochemical signals have been implicated as upstream regulators of YAP and TAZ, with multiple pro-fibrotic stimuli including matrix stiffness, TGFβ/SMAD, MRTF/SRF, and WNT (Ref. 5, 23, 24) signaling all potentially involved.

G protein coupled receptors are linked to effector proteins from four main classes of G-proteins (e.g., Gα_(12/13), Gα_(q/11), Gα_(i/o) or Gα_(s)). In some instances, G protein coupled receptor stimulates YAP/TAZ nuclear translocation and transcriptional activity. In other instances, G protein coupled receptors inhibit YAP/TAZ nuclear localization and activity via elevation of cAMP (see, e.g., FIG. 1, 2).

In some embodiments, activation (agonism) of a G protein coupled receptor results in YAP/TAZ hyper phosphorylation and inactivation under physiological conditions (e.g., agonism of the receptor prevents YAP/TAZ nuclear localization). This is in contrast to inactivation (antagonism) of G protein coupled receptor, which stimulates YAP/TAZ nuclear translocation and transcriptional activity, which results in expression of profibrotic genes, such as Acta2 (αSMA), Ctgf (Connective tissue growth factor), Fn1 (Fibronectin), Col1a1 (Collagen I), and Col1a2 (Collagen II).

In some embodiments, the present disclosure provides a method of agonizing a G protein coupled receptor in a cell, the method comprising contacting the cell with any one of compounds described herein, or a pharmaceutically acceptable salt thereof. In such embodiments, the compound selectively agonizes the Gα_(s) receptor (e.g., the compound is 100-fold, 50-fold, or 10-fold selective to Gα_(s) protein coupled receptor as compared to Gα_(12/13), Gα_(q/11) or Gα_(i/o) protein coupled receptor, or any combination of the aforementioned).

In some embodiments, the cell is a mesenchymal cell (e.g., the G protein coupled receptor is expressed in a mesenchymal cell). In some embodiments, the mesenchymal cell is a fibroblast (e.g., pulmonary, cardiac, hepatic, renal or dermal fibroblast) or a stellate cell (e.g., pancreatic stellate cell, hepatic stellate cell, podocyte, or osteocyte). In some embodiments, the G protein coupled receptor is preferentially expressed in mesenchymal cells as compared to epithelial or endothelial cells of a tissue (e.g., lung tissue or liver tissue). In one example, the G protein coupled receptor is preferentially expressed in pulmonary fibroblasts over alveolar epithelial cells. In another example, the G protein coupled receptor is preferentially expressed in hepatic stellate cells over hepatocytes.

In some embodiments, the G protein coupled receptor is Gα_(S) receptor. In one example, Gα_(S) receptor is expressed preferentially in the mesenchymal cell. In some embodiments, the Gα_(s) protein coupled receptor is a dopamine receptor (e.g., D1, D2, D3, D4, or D5 dopamine receptor). In some embodiments the dopamine receptor is a dopamine receptor D1 (DRD1). In one example, the methods of the present disclosure include selectively agonizing the dopamine receptor D1 (e.g., the compound of Formula (I) is 100-fold, 50-fold, or 10-fold selective to D1 dopamine receptor as compared to D2, D3, D4, or D5 receptor, or any combination of the aforementioned).

Referring to FIGS. 1 and 2, without being bound by a theory, it is believed that agonism of a G protein coupled receptor results in YAP/TAZ phosphorylation and subsequent degradation of YAP/TAZ in the cell. In some embodiments, the YAP/TAZ phosphorylation comprises phosphorylation of YAP serine 127. In some embodiments, the YAP/TAZ phosphorylation comprises phosphorylation of TAZ serine 89. In some embodiments, the YAP/TAZ phosphorylation comprises phosphorylation of YAP serine 127 and phosphorylation of TAZ serine 89. Hence, in some embodiments, the present disclosure provides a method of promoting YAP phosphorylation in a cell, the method comprising contacting the cell with any one of compounds described herein, or a pharmaceutically acceptable salt thereof. Because the compounds of the present disclosure promote degradation of the YAP/TAZ protein complex, in some embodiments, the present disclosure provides a method of reducing nuclear localization of YAP/TAZ in a cell, the method comprising contacting the cell with any one of the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof. Hence, the compounds of this disclosure render YAP/TAZ unable to perform its cellular function. In some embodiments, the present disclosure provides a method of inhibiting YAP/TAZ function in a cell, the method comprising contacting the cell with any one of the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof. Examples of the YAP/TAZ cellular functions include expression of profibrotic genes in the cell and production of fibrotic biomolecules (e.g., actin, collagen) by the cell. Generally, these fibrotic biomolecules constitute extra-cellular matrix surrounding the cell. Suitable examples of profibrotic genes include Acta2 (αSMA), Ctgf (Connective tissue growth factor), Fn1 (Fibronectin), Col1a1 (Collagen I), and Col1a2 (Collagen II). In some embodiments, the present disclosure provides a method of inhibiting expression of α-smooth muscle actin (αSMA) in a cell, the method comprising contacting the cell with any one of the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof. Concominantly, in some embodiments, the present disclosure provides a method of inhibiting production and deposition of extra-cellular matrix by a cell, the method comprising contacting the cell with any one of the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof. In some embodiments, inhibiting YAP/TAZ function in a cell by the compound of the present disclosure results in a prevention of accumulation of extracellular matrix in a tissue.

Without being bound by a theory, it is believed that agonism of a G protein coupled receptor reverses fiber formation and extracellular matrix accumulation (e.g., a G protein coupled receptor agonism leads to removing the fiber and extracellular matrix from a tissue). Hence, in some embodiments, the present disclosure provides a method enhancing extra-cellular matrix degradation by a cell, the method comprising contacting the cell with any one of the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof.

In some embodiments, the fiber formation, or fibrosis, is induced in a tissue by trauma or tissue injury. Normally, cells generate just the right amount of tissue to replace old tissue or repair tissue damage. Excessive connective tissue generation (e.g., in response to trauma or injury) results in pathological accumulation of fibrotic tissue (e.g., extracellular matrix proteins) leading to organ or tissue thickening and scarring.

In some embodiments, the present disclosure provides a method of treating or preventing a fibrotic pathology in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of any one of the compounds described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject in need of treatment of fibrotic pathology is diagnosed with fibrotic pathology by a treating physician.

In some embodiments, the fibrotic pathology is interstitial lung disease (ILD). In some embodiments, fibrotic pathology is lung tissue fibrosis, e.g., pulmonary fibrosis (PF) or idiopathic pulmonary fibrosis (IPF). Despite the name, cystic fibrosis is not considered an interstitial lung disease or predominantly a fibrotic pathology. Cystic fibrosis results from impaired ion transport, mucus dysfunction, and failure to effectively clear pathogens from the airways, which eventually results in scarring of the airways and lungs.

In some embodiments, fibrotic pathology is a liver tissue fibrosis, e.g., cirrhosis or biliary atresia. In some embodiments, fibrotic pathology is a heart tissue fibroses (cardiac fibrosis), e.g., atrial fibrosis, endomyocardial fibrosis, or post-myocardial infarction scarring. In some embodiments, fibrotic pathology is a brain tissue fibrosis, e.g., glial scar. In some embodiments, fibrotic pathology is arterial stiffness, arthrofibrosis (knee, shoulder, elbow, or other joints), kidney fibrosis (e.g., chronic kidney disease and fibrosis), liver fibrosis, nonalcoholic fatty liver, nonalcoholic steatohepatitis, Crohn's disease (intestinal scarring), Dupuytren's contracture (scar tissue in hands or fingers), skin tissue fibrosis, e.g., keloid (a scar on the skin), mediastinal fibrosis (soft tissue of the mediastinum), Peyronie's disease (scar in a penial tissue), nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis (scar on the soft tissue of the retroperitoneum) or adhesive capsulitis.

In some embodiments, the subject in need of prevention of fibrotic pathology is diagnosed with tissue trauma or injury by a treating physician. Suitable examples of tissue injury include injury caused by inhaled substances (e.g., silica or asbestos), drug-induced injury (injury caused by an antibiotic or an anticancer drug), tissue injury caused by autoimmune disease (e.g., rheumatoid arthritis, sclerosis, such as systemic sclerosis, lupus), injury caused by infection (e.g., tuberculosis, pneumonia, respiratory virus), or sarcoidosis.

Exemplary Therapeutic Compounds

Compounds of Formula (I)

In some embodiments, the present disclosure provides a compound of Formula

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 to 5 heteroatoms selected from N, O, and S, and 3-10-membered heterocycloalkyl ring comprising 1 to 3 heteroatoms independently selected from N, O, and S;

wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²;

each R² is independently selected from halo, OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino; and

R³ is selected from H and halo.

In some embodiments, the present disclosure provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, and 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S;

wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²; and

each R² is independently selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R¹ is 5-6-membered heteroaryl ring comprising 1 to 5 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 5-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 6-membered heteroaryl ring comprising 1 or 2 N atoms, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from pyridinyl, pyrimidinyl, pyrazinyl, diazinyl, triazinyl, tetrazinyl, and pentazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from pyridinyl, pyrimidinyl, and pyrazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is pyridinyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is 3-10-membered heterocycloalkyl ring comprising 1 to 3 heteroatoms independently selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 3-membered heterocycloalkyl ring comprising 1 heteroatom selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include aziridinyl and oxiranyl.

In some embodiments, R¹ is 4-membered heterocycloalkyl ring comprising 1 heteroatom selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include oxetanyl and azetidinyl.

In some embodiments, R¹ is 5-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include tetrahydrofuranyl, pyrrolidinyl, isoxazolidinyl, imidazolidinyl, and thiazolidinyl.

In some embodiments, R¹ is 6-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include morpholinyl, thiomorpholinyl, tetrahydropyranyl, piperazinyl, and piperidinyl.

In some embodiments, R¹ is 7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 8-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 9-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 10-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, pyrrolidinyl, pyranyl, morpholinyl, oxazinyl, dioxanyl, dioxinyl, diazinanyl, triazinanyl, trioxanyl, azepanyl, azepinyl, oxepanyl, oxepinyl, diazepanyl, diazepinyl, azocanyl, azocinyl, oxocanyl, oxocinyl, azonanyl, azoninyl, oxonanyl, and oxoninyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, and pyrrolidinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl and piperidinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is tetrahydropyranyl, optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is piperidinyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is selected from HO—C₁₋₆ alkyl and NH₂—C₁₋₆ alkyl.

In some embodiments, R¹ is HO—C₁₋₆ alkyl.

In some embodiments, R¹ is NH₂—C₁₋₆ alkyl.

In some embodiments, R² is independently selected from halo, OH, C₁₋₃ alkoxy, C₁₋₃ alkyl, and C₁₋₃ haloalkyl.

In some embodiments, R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from OH and NH₂. In some embodiments, R² is OH. In some embodiments, R² is NH₂. In some embodiments, R² is C₁₋₃ alkyl. In some embodiments, R² is HO—C₁₋₃ alkyl. In some embodiments, R² is NH₂—C₁₋₃ alkyl.

In some embodiments, R³ is H.

In some embodiments, R³ is halo.

In some embodiments, R³ is selected from Cl, F, and Br. In some embodiments, R³ is Cl. In some embodiments, R³ is F. In some embodiments, R³ is Br.

In some embodiments, the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

Compounds of Formula (II)

In some embodiments, the present disclosure provides a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from H and C₁₋₃ alkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, or di(C₁₋₃ alkyl)amino;

R², R³, and R⁴ are each independently selected from H, OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino; and

R⁵ is selected from H and halo.

In some embodiments of the compound of Formula (II), when R⁵ is H:

(i) at least one of R², R³, and R⁴ is not H;

(ii) if R² is H and R³ is OH, then R⁴ is not H or OH; and

(iii) if R² is OH, then at least one of R³ and R⁴ is not H.

In some embodiments, the present disclosure provides a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from H and C₁₋₃ alkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, or di(C₁₋₃ alkyl)amino; and

R², R³, and R⁴ are each independently selected from H, OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments of the compound of Formula (II), at least one of R², R³, and R⁴ is not H. In some embodiments of the compound of Formula (II), if R² is H and R³ is OH, then R⁴ is not H or OH. In some embodiments of the compound of Formula (II), if R² is OH, then at least one of R³ and R⁴ is not H.

In some embodiments of the compound of Formula (II):

(i) at least one of R², R³, and R⁴ is not H;

(ii) if R² is H and R³ is OH, then R⁴ is not H or OH; and

(iii) if R² is OH, then at least one of R³ and R⁴ is not H.

In some embodiments, R¹ is H.

In some embodiments, R¹ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl).

In some embodiments, R¹ is selected from HO—C₁₋₃ alkyl and NH₂—C₁₋₃ alkyl. In some embodiments, R¹ is HO—C₁₋₃ alkyl. In some embodiments, R¹ is NH₂—C₁₋₃ alkyl.

In some embodiments, at least one of R², R³, and R⁴ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, at least one of R², R³, and R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments, at least one of R², R³, and R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments, at least one of R², R³, and R⁴ is C₁₋₃ alkyl.

In some embodiments, R² is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R² is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R² is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R² is HO—C₁₋₃ alkyl. In some embodiments, R² is NH₂—C₁₋₃ alkyl. In some embodiments, R² is OH. In some embodiments, R² is NH₂. In some embodiments, R² is H.

In some embodiments, R³ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R³ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R³ is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R³ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R³ is HO—C₁₋₃ alkyl. In some embodiments, R³ is NH₂—C₁₋₃ alkyl. In some embodiments, R³ is OH. In some embodiments, R³ is NH₂. In some embodiments, R³ is H.

In some embodiments, R⁴ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R⁴ is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R⁴ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R⁴ is HO—C₁₋₃ alkyl. In some embodiments, R⁴ is NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is OH. In some embodiments, R⁴ is NH₂. In some embodiments, R⁴ is H.

In some embodiments:

R³ is OH; and

R² is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments:

R³ is OH; and

R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments:

R³ is OH; and

R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments:

R³ is OH; and

R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments:

R⁴ is OH; and

R³ is selected from H, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments:

R⁴ is OH; and

R³ is selected from H, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments:

R² is OH; and at least one of R³ and R⁴ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments:

R² is OH; and at least one of R³ and R⁴ is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments, R³ is OH and R² is C₁₋₃ alkyl. In some embodiments, R³ is C₁₋₃ alkyl and R² is OH. In some embodiments, R³ is OH and R⁴ is C₁₋₃ alkyl. In some embodiments, R³ is C₁₋₃ alkyl and R⁴ is OH.

In some embodiments:

R⁵ is halo; and

R², R³, and R⁴ are each independently selected from H, OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R², R³, and R⁴ are each independently selected from H, OH, and C₁₋₃ alkyl. In some embodiments, R², R³, and R⁴ are each H.

In some embodiments, R⁵ is H. In some embodiments, R⁵ is halo. In some embodiments, R⁵ is selected from Cl, Br, and F. In some embodiments, R⁵ is Cl. In some embodiments, R⁵ is Br. In some embodiments, R⁵ is F.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (II) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

Compounds of Formula (II)

In some embodiments, the present disclosure provides a compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is selected from CH₂ and O;

R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 to 5 heteroatoms selected from N, O, and S, and 3-10-membered heterocycloalkyl ring comprising 1 to 3 heteroatoms independently selected from N, O, and S;

wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²;

each R² is independently selected from halo, OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino;

R³ is selected from H and halo; and

R⁴ is selected from H and C₁₋₃ alkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, or di(C₁₋₃ alkyl)amino.

In some embodiments, X¹ is CH₂. In some embodiments, X¹ is O.

In some embodiments, R³ is H. In some embodiments, R³ is halo. In some embodiments, R³ is selected from Cl, Br, and F. In some embodiments, R³ is Cl. In some embodiments, R³ is Br. In some embodiments, R³ is F.

In some embodiments, R⁴ is selected from H and C₁₋₃ alkyl.

In some embodiments, R⁴ is H. In some embodiments, R⁴ is C₁₋₃ alkyl.

In some embodiments, R¹ is 5-6-membered heteroaryl ring comprising 1 to 5 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is 5-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is selected from thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 6-membered heteroaryl ring comprising 1 or 2 N atoms, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from pyridinyl, pyrimidinyl, pyrazinyl, diazinyl, triazinyl, tetrazinyl, and pentazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is selected from pyridinyl, pyrimidinyl, and pyrazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is pyridinyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, the compound of Formula (III) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (III) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is 3-10-membered heterocycloalkyl ring comprising 1 to 3 heteroatoms independently selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is 3-membered heterocycloalkyl ring comprising 1 heteroatom selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include aziridinyl and oxiranyl.

In some embodiments, R¹ is 4-membered heterocycloalkyl ring comprising 1 heteroatom selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include oxetanyl and azetidinyl.

In some embodiments, R¹ is 5-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R. Examples of such rings include tetrahydrofuranyl, pyrrolidinyl, isoxazolidinyl, imidazolidinyl, and thiazolidinyl.

In some embodiments, R¹ is 6-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R². Examples of such rings include morpholinyl, thiomorpholinyl, tetrahydropyranyl, piperazinyl, and piperidinyl.

In some embodiments, R¹ is 7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R.

In some embodiments, R¹ is 8-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 9-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is 10-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, pyrrolidinyl, pyranyl, morpholinyl, oxazinyl, dioxanyl, dioxinyl, diazinanyl, triazinanyl, trioxanyl, azepanyl, azepinyl, oxepanyl, oxepinyl, diazepanyl, diazepinyl, azocanyl, azocinyl, oxocanyl, oxocinyl, azonanyl, azoninyl, oxonanyl, and oxoninyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, and pyrrolidinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is selected from tetrahydropyranyl and piperidinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

In some embodiments, R¹ is tetrahydropyranyl, optionally substituted with 1, 2, or 3 substituents independently selected from R². In some embodiments, R¹ is any one of R¹ described herein for Formula (I).

In some embodiments, the compound of Formula (III) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula (III) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is selected from HO—C₁₋₆ alkyl and NH₂—C₁₋₆ alkyl.

In some embodiments, R¹ is HO—C₁₋₆ alkyl.

In some embodiments, R¹ is NH₂—C₁₋₆ alkyl.

In some embodiments, each R² is independently selected from halo, OH, C₁₋₃ alkoxy, C₁₋₃ alkyl, and C₁₋₃ haloalkyl. In some embodiments, R² is any of the R² groups described herein for the compound of Formula (I). In some embodiments, R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from OH and NH₂. In some embodiments, R² is OH. In some embodiments, R² is NH₂. In some embodiments, R² is C₁₋₃ alkyl. In some embodiments, R² is HO—C₁₋₃ alkyl. In some embodiments, R² is NH₂—C₁₋₃ alkyl.

In some embodiments, the compound of Formula (III) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

Compounds of Formula (IV)

In some embodiments, the present disclosure provides a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

X¹ is selected from CH₂ and 0;

X² is selected from CR³ and N;

R¹ is selected from H and C₁₋₃ alkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, or di(C₁₋₃ alkyl)amino;

R⁵ is selected from H and halo; and

R², R³, and R⁴ are each independently selected from H, OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments of the compound of Formula (IV), if R⁵ is H and X² is CR³, then at least one of R², R³, and R⁴ is not H.

In some embodiments, X¹ is CH₂. In some embodiments, X¹ is O.

In some embodiments, R¹ is selected from H and C₁₋₃ alkyl.

In some embodiments, R¹ is H. In some embodiments, R¹ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R¹ is selected from HO—C₁₋₃ alkyl and NH₂—C₁₋₃ alkyl. In some embodiments, R¹ is HO—C₁₋₃ alkyl. In some embodiments, R¹ is NH₂—C₁₋₃ alkyl.

In some embodiments, R⁵ is H. In some embodiments, R⁵ is halo. In some embodiments, R⁵ is selected from Cl, Br, and F. In some embodiments, R⁵ is Cl. In some embodiments, R⁵ is Br. In some embodiments, R⁵ is F.

In some embodiments, X² is N. In some embodiments, X² is CR³.

In some embodiments, at least one of R², R³, and R⁴ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, at least one of R², R³, and R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

In some embodiments, at least one of R², R³, and R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

In some embodiments, at least one of R², R³, and R⁴ is C₁₋₃ alkyl.

In some embodiments, R² is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R² is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R² is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R² is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R² is HO—C₁₋₃ alkyl. In some embodiments, R² is NH₂—C₁₋₃ alkyl. In some embodiments, R² is OH. In some embodiments, R² is NH₂. In some embodiments, R² is H.

In some embodiments, R³ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R³ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R³ is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R³ is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R³ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R³ is HO—C₁₋₃ alkyl. In some embodiments, R³ is NH₂—C₁₋₃ alkyl. In some embodiments, R³ is OH. In some embodiments, R³ is NH₂. In some embodiments, R³ is H.

In some embodiments, R⁴ is selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R⁴ is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is selected from C₁₋₃ alkyl and HO—C₁₋₃ alkyl. In some embodiments, R⁴ is C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl). In some embodiments, R⁴ is HO—C₁₋₃ alkyl. In some embodiments, R⁴ is NH₂—C₁₋₃ alkyl. In some embodiments, R⁴ is OH. In some embodiments, R⁴ is NH₂. In some embodiments, R⁴ is H.

In some embodiments, the compound of Formula (IV) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments:

R⁵ is halo; and

R², R³, and R⁴ are each independently selected from H, OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R², R³, and R⁴ are each independently selected from H, OH, and C₁₋₃ alkyl. In some embodiments, R², R³, and R⁴ are each H.

In some embodiments:

R³ is H;

R⁵ is H; and

R² and R⁴ are each independently selected from OH and C₁₋₃ alkyl.

In some embodiments, the compound of Formula (IV) has formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, R² and R⁴ are each independently selected from H, halo, OH, C₁₋₃ alkoxy, C₁₋₃ alkyl, and C₁₋₃ haloalkyl.

In some embodiments, the compound of Formula (IV) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the the compound of any one of the foregoing Formulae is hydrophilic. In such embodiments, the structure of the compound contains hydrogen bond donor (HBD) atoms that are capable of forming hydrogen bonds with molecules of water and with the amino acids within the active site of a G protein coupled receptor. In some embodiments, the compound of any one of the foregoing Formulae contains at least 2, 3, 4, 5, or 6 HBD atoms (e.g., heteroatoms such as O, N or S). In some embodiments, the compound of any one of the foregoing Formulae contains at least one hydroxyl group (e.g., 1, 2, 3, 4, 5, or 6 hydroxyl groups). In some embodiments, the compound of any one of the foregoing Formulae contains amino groups (e.g., 1, 2, 3, 4, 5, or 6 amino groups).

In some embodiments, the compound of any one of the foregoing Formulae does not penetrate the blood brain barrier or only an insignificant amount of the compound of any one of the foregoing Formulae penetrates the blood brain barrier after the compound is administered to a subject (e.g., not more than about 0.1 wt. %, about 1 wt. %, about 5 wt. %, about 10 wt. %, or about 20 wt. % of the amount of the compound administered to the subject penetrates the blood brain barrier). In one example, the compound of any one of the foregoing Formulae is ineffective or only weakly effective in treating central nervous system (CNS) disorders due to its hydrophilicity and subsequent inability to penetrate the blood bran barrier.

Pharmaceutical Compositions and Formulations

The present application also provides pharmaceutical compositions comprising an effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise at least one of any one of the additional therapeutic agents described. In certain embodiments, the application also provides pharmaceutical compositions and dosage forms comprising any one the additional therapeutic agents described herein (e.g., in a kit). The carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present application include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

The compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100% with the balance made up from the suitable pharmaceutically acceptable excipients. The contemplated compositions may contain 0.001%-100% of any one of the compounds and therapeutic agents provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%, wherein the balance may be made up of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.

Routes of Administration and Dosage Forms

The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration. Acceptable routes of administration include, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal.

Compositions and formulations described herein may conveniently be presented in a unit dosage form, e.g., tablets, capsules (e.g., hard or soft gelatin capsules), sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, Md. (20th ed. 2000). Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product.

In some embodiments, any one of the compounds and therapeutic agents disclosed herein are administered orally. Compositions of the present application suitable for oral administration may be presented as discrete units such as capsules, sachets, granules or tablets each containing a predetermined amount (e.g., effective amount) of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non-aqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption. In the case of tablets for oral use, carriers that are commonly used include lactose, sucrose, glucose, mannitol, and silicic acid and starches. Other acceptable excipients may include: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or infusion solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of the present application may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include cocoa butter, beeswax, and polyethylene glycols.

The pharmaceutical compositions of the present application may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, U.S. Pat. No. 6,803,031. Additional formulations and methods for intranasal administration are found in Ilium, L., J Pharm Pharmacol, 56:3-17, 2004 and Ilium, L., Eur J Pharm Sci 11:1-18, 2000.

The topical compositions of the present disclosure can be prepared and used in the form of an aerosol spray, cream, emulsion, solid, liquid, dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder, patch, pomade, solution, pump spray, stick, towelette, soap, or other forms commonly employed in the art of topical administration and/or cosmetic and skin care formulation. The topical compositions can be in an emulsion form. Topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application. In some embodiments, the topical composition comprises a combination of any one of the compounds and therapeutic agents disclosed herein, and one or more additional ingredients, carriers, excipients, or diluents including absorbents, anti-irritants, anti-acne agents, preservatives, antioxidants, coloring agents/pigments, emollients (moisturizers), emulsifiers, film-forming/holding agents, fragrances, leave-on exfoliants, prescription drugs, preservatives, scrub agents, silicones, skin-identical/repairing agents, slip agents, sunscreen actives, surfactants/detergent cleansing agents, penetration enhancers, and thickeners.

The compounds and therapeutic agents of the present application may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition. Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein.

According to another embodiment, the present application provides an implantable drug release device impregnated with or containing a compound or a therapeutic agent, or a composition comprising a compound of the present application or a therapeutic agent, such that said compound or therapeutic agent is released from said device and is therapeutically active.

Dosages and Regimens

In the pharmaceutical compositions of the present application, a therapeutic compound is present in an effective amount (e.g., a therapeutically effective amount). Effective doses may vary, depending on the diseases treated, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.

In some embodiments, an effective amount of a therapeutic compound can range, for example, from about 0.001 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kg to about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about 0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg; from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about 0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0.1 mg/kg to about 200 mg/kg; from about 0.1 mg/kg to about 150 mg/kg; from about 0.1 mg/kg to about 100 mg/kg; from about 0.1 mg/kg to about 50 mg/kg; from about 0.1 mg/kg to about 10 mg/kg; from about 0.1 mg/kg to about 5 mg/kg; from about 0.1 mg/kg to about 2 mg/kg; from about 0.1 mg/kg to about 1 mg/kg; or from about 0.1 mg/kg to about 0.5 mg/kg).

In some embodiments, an effective amount of a therapeutic compound is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 5 mg/kg.

The foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or non-daily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month). The compounds and compositions described herein can be administered to the subject in any order. A first therapeutic agent, such as a compound of the present disclosure, can be administered prior to or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before or after), or concomitantly with the administration of a second therapeutic agent, such as an anti-fibrotic agent described herein, to a subject in need of treatment. Thus, the compound of the present disclosure, or a composition containing the compound, can be administered separately, sequentially or simultaneously with the second therapeutic agent, such as an anti-fibrotic agent described herein. When the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a second or third therapeutic agent are administered to the subject simultaneously, the therapeutic agents may be administered in a single dosage form (e.g., tablet, capsule, or a solution for injection or infusion).

In some embodiments, the second (additional) therapeutic agent is a drug that is useful in treating or preventing a fibrotic pathology. Suitable examples of such drugs include nintedanib, pirfenidone, or prednisone, or immunosuppressants, such as cyclophosphamide, azathioprine, methotrexate, penicillamine, and cyclosporine.

In some embodiments, the additional therapeutic agent is dopamine, or a pharmaceutically acceptable salt thereof.

In some embodiments, the additional therapeutic agent is a dopamine receptor agonist. In some embodiments, the dopamine receptor agonist is selected from: ABT-413, A-86929, dihydrexidine (DHX), dinapsoline, dinoxyline, doxanthrine, SKF-81297, SKF-82958, SKF-38393, fenoldopam, 6-Br-APB, stepholidine, A-68930, A-77636, CY-208-243, SKF-89145, SKF-89626, 7,8-dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline, cabergoline, pergolide, R(−)-2,10,11-trihydroxyaporphine, (R)-(−)-apomorphine, R(−)-propylnorapomorphine, R(+)-6-bromo-APB, R(−)-2,10,11-trihydroxy-N-propyl-noraporphine, 6,7-ADTN, mesulergine, N-methyldopamine, 4-hydroxyphenethylamine, cabergoline, 3-hydroxyphenethylamine, pramipexole, PD-168077, fenoldopam, (±)-PD 128-907, (+)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin, bromocriptine, ropinirole, LY-163-502, dipropyldopamine, B-HT 920, piribedil, (+)-UH 232, pergolide, (−)-quinpirole, R(−)-2,11-dihydroxy-10-methoxyapomorphine, or a pharmaceutically acceptable salt thereof.

In some embodiments, the second (additional) therapeutic agent is an anti-inflammatory drug. Suitable examples of such drugs include NSAIDs such as celecoxib, rofecoxib, ibuprofen, naproxen, aspirin, diclofenac, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, methotrexate, BAY 11-7082, or a pharmaceutically acceptable salt thereof. Suitable examples of steroid anti-inflammatory agents include cortisol, corticosterone, hydrocortisone, aldosterone, deoxycorticosterone, triamcinolone, bardoxolone, bardoxolone methyl, triamcinolone, cortisone, prednisone, and methylprednisolone, or a pharmaceutically acceptable salt thereof.

Kits

The present invention also includes pharmaceutical kits useful, for example, in the treatment of disorders, diseases and conditions referred to herein, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

Definitions

As used herein, the term “about” means “approximately” (e.g., plus or minus approximately 10% of the indicated value).

As used herein, the term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures named or depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

The terms “pharmaceutical” and “pharmaceutically acceptable” are employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal. In some embodiments, the cell is a mesenchymal cell. In some embodiments, the cell is a fibroblast (e.g., cardiac, dermal or lung fibroblast). In some embodiments, the cell is a hepatic stellate cell.

As used herein, the term “contacting” refers to the bringing together of indicated moieties or items in an in vitro system, an ex vivo system, or an in vivo system. For example, “contacting” a cell with a compound provided herein includes the act of administering that compound to a mammal (e.g., a human) containing that cell as well as, for example, introducing that compound into a cell culture containing that cell.

As used herein, the term “mammal” includes, without limitation, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, elephants, deer, non-human primates (e.g., monkeys and apes), house pets, and humans.

As used herein, the phrase “effective amount” or “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, mammal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician.

As used herein the term “treating” or “treatment” refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).

As used herein, the term “preventing” or “prevention” of a disease, condition or disorder refers to decreasing the risk of occurrence of the disease, condition or disorder in a subject or group of subjects (e.g., a subject or group of subjects predisposed to or susceptible to the disease, condition or disorder). In some embodiments, preventing a disease, condition or disorder refers to decreasing the possibility of acquiring the disease, condition or disorder and/or its associated symptoms. In some embodiments, preventing a disease, condition or disorder refers to completely or almost completely stopping the disease, condition or disorder from occurring.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. In some embodiments, the compound is a pharmaceutically acceptable acid addition salt. In some embodiments, acids commonly employed to form pharmaceutically acceptable salts of the therapeutic compounds described herein include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

In some embodiments, bases commonly employed to form pharmaceutically acceptable salts of the therapeutic compounds described herein include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH—(C1-C6)-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like.

As used herein, the phrase “optionally substituted” means unsubstituted or substituted. The substituents are independently selected, and substitution can be at any chemically accessible position. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms. It is to be understood that substitution at a given atom is limited by valency.

Throughout the definitions, the term “C_(n-m)” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C₁₋₄, C₁₋₆, and the like.

As used herein, the term “C_(n-m) alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, without limitation, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, the term “C_(n-m) haloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms that may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “amino” refers to a group of formula —NH₂.

As used herein, the term “C_(n-m) alkylamino” refers to a group of formula —NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Suitable examples of alkylamino groups include N-methylamino, N-ethylamino, N-propylamino (e.g., N-(n-propyl)amino and N-isopropylamino), N-butylamino (e.g., N-(n-butyl)amino and N-(tert-butyl)amino), and the like.

As used herein, the term “di C_(n-m) alkylamino” refers to a group of formula —N(alkyl)₂, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Suitable examples of dialkylamino groups include N,N-methylehtylamino, N,N-diethylamino, N,N-propylethylamino, N,N-butylisopropylamino, and the like.

As used herein, the term “C_(n-m) alkoxy”, employed alone or in combination with other terms, refers to a group of formula —O-alkyl, wherein the alkyl group has n to m carbons. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “HO—C₁₋₃ alkyl” refers to a group of formula —(C₁₋₃ alkylene)-OH. As used herein, the term “NH₂—C₁₋₃ alkyl” refers to a group of formula —(C₁₋₃ alkylene)-NH₂.

As used herein, “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br.

As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5-6 monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. A five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered ring heteroaryls include, without limitation, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six-membered ring heteroaryls include, without limitation, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include, without limitation, pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydropyran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by 1 or 2 independently selected oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)₂, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

EXAMPLES

Materials and Methods

Cell culture: Cells were all maintained in EMEM (ATCC) containing 10% FBS, unless otherwise noted. IMR-90 embryonic lung fibroblasts and NIH-3T3 mouse fibroblasts were purchased from ATCC. Doxycycline-inducible Tet-On NIH3T3 expressing TAZ4SA or control empty vector were described previously. Normal Human Alveolar Epithelial Cells (NHAEpCs), Normal Human Microvascular Endothelial Cells (NHMVECs), Normal Human Lung Fibroblasts (NHLFs), and Human Dermal Fibroblasts (HDFs) were purchased from Lonza and were cultured in the proprietary media per Lonza's recommendation. Human Adult Cardiac Fibroblasts (HACFs) and Hepatic Stellate Cells (HSCs) were purchased from ScienCell and were cultured in the proprietary media per ScienCell's recommendation. Hepatocytes were purchased from Samsara and were cultured in the proprietary media per Samsara's recommendation. All additional experiments with pulmonary fibroblasts used primary human lung fibroblasts isolated by explant culture from the lungs of subjects diagnosed with IPF who underwent lung transplantation, or donors whose organs were rejected for transplantation (non-IPF), generously provided by Peter Bitterman and Craig Henke at the University of Minnesota under a protocol approved by the University of Minnesota Institutional Review Board. All primary cell culture experiments were performed with cells at passage six or less.

Chemicals and Reagents: Dimethyl sulfoxide (DMSO), Y-27632, endothelin 1 (ET-1), and ascorbic acid were purchased from Sigma-Aldrich. Dihydrexidine (DHX), SKF-81297, fenoldopam, forskolin, and prostaglandin E2 were purchased from Tocris Bioscience. lysophosphatidic acid (LPA), and serotonin (5-HT) were purchased from Cayman Chemical. SCH 39166 was purchased from Santa Cruz Biotechnology. TGFβ1 was purchased from eBioscience.

GPCRome Profiling and qPCR: GPCRome profiling was performed according to the manufacturer's suggestions (Qiagen). Cells were grown in their recommended growth media for 24 hours prior to RNA isolation using RNeasy Plus Mini Kit (Qiagen) according to manufacturer's instructions. Isolated RNA (1000 ng) was then used to synthesize cDNA using the RT² First Strand Kit (Qiagen) and the G Protein Coupled Receptors 384HT PCR Array was analyzed using a LightCycler 480 (Roche). Data are shown as 1/Ct (FIG. 3), GAPDH for fibroblast and epithelial cell datasets were nearly identical (17.39 and 17.41 respectively). Raw Ct values for all receptors are available (Table 1).

TABLE 1 Receptors marked (*) were identified to exclusively couple to Gα_(s) (See e.g., Ref. 21) Fibro- Aveolar blast Epithelial Uni Gene Gen Bank Symbol Description cT cT Hs.377783 NM_001118 ADCYAP1R1 Adenylate cyclase activating polypeptide 1 34.98 34.41 (pituitary) receptor type I Hs.77867 NM_000674 ADORA1 Adenosine A1 receptor 29.7 27.95 Hs.197029 NM_000675 ADORA2A Adenosine A2a receptor 32.49 30.97 Hs.167046 NM_000676 ADORA2B Adenosine A2b receptor 26.82 26.08 Hs.281342 NM_000677 ADORA3 Adenosine A3 receptor 37.78 35.78 Hs.709175 NM_033303 ADRA1A Adrenergic, alpha-1A-, receptor 34.69 33.45 Hs.368632 NM_000679 ADRA1B Adrenergic, alpha-1B-, receptor 32.9 29.79 Hs.557 NM_000678 ADRA1D Adrenergic, alpha-1D-, receptor 29.16 32.89 Hs.249159 NM_000681 ADRA2A Adrenergic, alpha-2A-, receptor 33.65 34.51 Hs.247686 NM_000682 ADRA2B Adrenergic, alpha-2B-, receptor 36.07 40 Hs.123022 NM_000683 ADRA2C Adrenergic, alpha-2C-, receptor 33.79 33.89 Hs.99913 NM_000684 ADRB1 Adrenergic, beta-1-, receptor 36.08 30.93 Hs.591251 NM_000024 ADRB2 Adrenergic, beta-2-, receptor, surface 24.66 22.13 Hs.2549 NM_000025 ADRB3 Adrenergic, beta-3-, receptor 33.1 34.93 Hs.728754 NM_031850 AGTR1 Angiotensin II receptor, type 1 26.71 31.48 Hs.405348 NM_000686 AGTR2 Angiotensin II receptor, type 2 35.14 40 Hs.438311 NM_005161 APLNR Apelin receptor 36.26 36.72 Hs.2131 NM_000706 AVPR1A Arginine vasopressin receptor 1A 33.8 32.59 Hs.1372 NM_000707 AVPR1B Arginine vasopressin receptor 1B 30.85 27.69 Hs.567240 NM_000054 AVPR2 Arginine vasopressin receptor 2 (*) 35.1 39 Hs.194654 NM_001702 BAI1 Brain-specific angiogenesis inhibitor 1 35.5 31.24 Hs.524138 NM_001703 BAI2 Brain-specific angiogenesis inhibitor 2 28.05 31.49 Hs.13261 NM_001704 BAI3 Brain-specific angiogenesis inhibitor 3 34.63 35.72 Hs.525572 NM_000710 BDKRB1 Bradykinin receptor B1 25.52 31.86 Hs.654542 NM_000623 BDKRB2 Bradykinin receptor B2 29.25 32.77 Hs.121484 NM_001727 BRS3 Bombesin-like receptor 3 34.59 34.82 Hs.591148 NM_004054 C3AR1 Complement component 3a receptor 1 31.75 31.58 Hs.2161 NM_001736 C5AR1 Complement component 5a receptor 1 30.59 30.75 Hs.489127 NM_001742 CALCR CALCITONIN RECEPTOR 38.19 37.02 Hs.470882 NM_005795 CALCRL Calcitonin receptor-like 31.19 28.81 Hs.435615 NM_000388 CASR Calcium-sensing receptor 31.32 31.84 Hs.146346 NM_001296 CCBP2 Chemokine binding protein 2 27.74 27.65 Hs.129 NM_000730 CCKAR Cholecystokinin A receptor 32.43 36.62 Hs.203 NM_176875 CCKBR Cholecystokinin B receptor 34.99 39.34 Hs.301921 NM_001295 CCR1 Chemokine (C-C motif) receptor 1 30.53 30.92 Hs.278446 NM_016602 CCR10 Chemokine (C-C motif) receptor 10 29.82 28.25 Hs.511794 NM_001123396 CCR2 Chemokine (C-C motif) receptor 2 34.75 33.51 Hs.506190 NM_001837 CCR3 Chemokine (C-C motif) receptor 3 33.35 36.14 Hs.184926 NM_005508 CCR4 Chemokine (C-C motif) receptor 4 32.99 34.95 Hs.450802 NM_000579 CCR5 Chemokine (C-C motif) receptor 5 32.86 32.49 Hs.46468 NM_004367 CCR6 Chemokine (C-C motif) receptor 6 33.77 30.9 Hs.370036 NM_001838 CCR7 Chemokine (C-C motif) receptor 7 30.45 32.7 Hs.113222 NM_005201 CCR8 Chemokine (C-C motif) receptor 8 36.9 40 Hs.225946 NM_006641 CCR9 Chemokine (C-C motif) receptor 9 33.71 35.12 Hs.729361 NM_016557 CCRL1 Chemokine (C-C motif) receptor-like 1 24.68 29.02 Hs.535713 NM_003965 CCRL2 Chemokine (C-C motif) receptor-like 2 32.82 26.46 Hs.466039 NM_001784 CD97 CD97 molecule 23.15 23.23 Hs.252387 NM_014246 CELSR1 Cadherin, EGF LAG seven-pass G-type 29.57 27.06 receptor 1 Hs.57652 NM_001408 CELSR2 Cadherin, EGF LAG seven-pass G-type 32.54 31.57 receptor 2 Hs.631926 NM_001407 CELSR3 Cadherin, EGF LAG seven-pass G-type 32.97 31.12 receptor 3 Hs.632119 NM_000738 CHRM1 Cholinergic receptor, muscarinic 1 40 35.56 Hs.535891 NM_000739 CHRM2 Cholinergic receptor, muscarinic 2 26.19 31.13 Hs.7138 NM_000740 CHRM3 Cholinergic receptor, muscarinic 3 36.75 36.74 Hs.248100 NM_000741 CHRM4 Cholinergic receptor, muscarinic 4 30.1 29.43 Hs.584747 NM_012125 CHRM5 Cholinergic receptor, muscarinic 5 31.48 31.18 Hs.197143 NM_004072 CMKLR1 CHEMOKINE-LIKE RECEPTOR 1 34.55 34.77 Hs.75110 NM_016083 CNR1 Cannabinoid receptor 1 (brain) 36.13 33.43 Hs.73037 NM_001841 CNR2 Cannabinoid receptor 2 (macrophage) 27.88 26.15 Hs.300684 NM_014478 CRCP CGRP receptor component 24.2 23.45 Hs.417628 NM_004382 CRHR1 Corticotropin releasing hormone receptor 1 33 32.61 Hs.729970 NM_001883 CRHR2 Corticotropin releasing hormone receptor 2 35.59 35.16 Hs.78913 NM_001337 CX3CR1 Chemokine (C-X3-C motif) receptor 1 35.81 36.67 Hs.194778 NM_000634 CXCR1 Chemokine (C-X-C motif) receptor 1 38.63 34.23 Hs.846 NM_001557 CXCR2 Chemokine (C-X-C motif) receptor 2 26.1 25.7 Hs.198252 NM_001504 CXCR3 Chemokine (C-X-C motif) receptor 3 31.54 30.31 Hs.593413 NM_003467 CXCR4 Chemokine (C-X-C motif) receptor 4 33.79 27.46 Hs.113916 NM_001716 CXCR5 Chemokine (C-X-C motif) receptor 5 34.73 31.79 Hs.34526 NM_006564 CXCR6 Chemokine (C-X-C motif) receptor 6 29.43 28.22 Hs.471751 NM_020311 CXCR7 Chemokine (C-X-C motif) receptor 7 29.5 29.02 Hs.201300 NM_006639 CYSLTR1 Cysteinyl leukotriene receptor 1 34.63 35.83 Hs.253706 NM_020377 CYSLTR2 Cysteinyl leukotriene receptor 2 36.85 35.32 Hs.153381 NM_002036 DARC Duffy blood group, chemokine receptor 40 37.48 Hs.2624 NM_000794 DRD1 Dopamine receptor D1 (*) 26.47 40 Hs.73893 NM_000795 DRD2 Dopamine receptor D2 32.97 30.29 Hs.121478 NM_000796 DRD3 Dopamine receptor D3 32.27 33.02 Hs.99922 NM_000797 DRD4 Dopamine receptor D4 33.5 33.22 Hs.380681 NM_000798 DRD5 Dopamine receptor D5 (*) 35.72 35 Hs.183713 NM_001957 EDNRA Endothelin receptor type A 24.05 28.15 Hs.82002 NM_000115 EDNRB Endothelin receptor type B 27.72 27.09 Hs.132314 NM_022159 ELID1 EGF, latrophilin and seven transmembrane 25.5 27.01 domain containing 1 Hs.2375 NM_001974 EMR1 Egf-like module containing, mucin-like, 31.03 32.47 hormone receptor-like 1 Hs.482562 NM_001992 F2R Coagulation factor II (thrombin) receptor 20.46 23.5 Hs.154299 NM_005242 F2RL1 Coagulation factor II (thrombin) receptor- 23.32 20.47 like 1 Hs.42502 NM_004101 F2RL2 Coagulation factor II (thrombin) receptor- 23.34 29.22 like 2 Hs.137574 NM_003950 F2RL3 Coagulation factor II (thrombin) receptor- 32.52 30.04 like 3 Hs.248127 NM_005303 FFAR1 Free fatty acid receptor 1 34.82 33.99 Hs.248056 NM_005306 FFAR2 Free fatty acid receptor 2 40 36.9 Hs.248055 NM_005304 FFAR3 Free fatty acid receptor 3 35.52 35.47 Hs.753 NM_002029 FPR1 Formyl peptide receptor 1 35.8 40 Hs.99855 NM_001462 FPR2 Formyl peptide receptor 2 33.2 34.11 Hs.445466 NM_002030 FPR3 Formyl peptide receptor 3 33.45 33.8 Hs.1428 NM_181446 FSHR Follicle stimulating hormone receptor 35.87 35.7 Hs.94234 NM_003505 FZD1 Frizzled family receptor 1 23.26 25.3 Hs.31664 NM_007197 FZD10 Frizzled family receptor 10 37.64 31.86 Hs.142912 NM_001466 FZD2 Frizzled family receptor 2 26.96 25.31 Hs.40735 NM_017412 FZD3 Frizzled family receptor 3 (*) 29.18 25.21 Hs.19545 NM_012193 FZD4 Frizzled family receptor 4 25.59 27.44 Hs.17631 NM_003468 FZD5 Frizzled family receptor 5 29.48 25.43 Hs.591863 NM_003506 FZD6 Frizzled family receptor 6 23.58 22.69 Hs.173859 NM_003507 FZD7 Frizzled family receptor 7 25.43 28.28 Hs.302634 NM_031866 FZD8 Frizzled family receptor 8 28.64 27.28 Hs.647029 NM_003508 FZD9 Frizzled family receptor 9 33.01 33.15 Hs.167017 NM_001470 GABBR1 Gamma-aminobutyric acid (GABA) B 29.81 30.5 receptor, 1 Hs.198612 NM_005458 GABBR2 Gamma-aminobutyric acid (GABA) B 23.5 29.12 receptor, 2 Hs.272191 NM_001480 GALR1 Galanin receptor 1 37.82 35.83 Hs.666366 NM_003857 GALR2 GALANIN RECEPTOR 2 34.97 33.03 Hs.158353 NM_003614 GALR3 Galanin receptor 3 35.06 40 Hs.208 NM_000160 GCGR Glucagon receptor (*) 40 33.78 Hs.767 NM_000823 GHRHR Growth hormone releasing hormone 32.27 32.5 receptor (*) Hs.248115 NM_004122 GHSR Growth hormone secretagogue receptor 35.58 33.62 Hs.658534 NM_000164 GIPR Gastric inhibitory polypeptide receptor (*) 31.28 29.57 Hs.389103 NM_002062 GLP1R Glucagon-like peptide 1 receptor 40 37.36 Hs.248202 NM_004246 GLP2R Glucagon-like peptide 2 receptor 35.47 37.79 Hs.407587 NM_000406 GNRHR Gonadotropin-releasing hormone receptor 33.11 32.34 Hs.160954 NM_170699 GPBAR1 G protein-coupled bile acid receptor 1 (*) 32.49 31.71 Hs.20961 NM_001505 GPER G protein-coupled estrogen receptor 1 28.52 28.89 Hs.184907 NM_005279 GPR1 G protein-coupled receptor 1 26.65 26.31 Hs.350569 NM_054021 GPR101 G protein-coupled receptor 101 (*) 34.32 35.07 Hs.256897 NM_153840 GPR110 G protein-coupled receptor 110 33.06 26.43 Hs.715357 NM_153839 GPR111 G protein-coupled receptor 111 33.75 29.59 Hs.381354 NM_153834 GPR112 G protein-coupled receptor 112 40 40 Hs.631878 NM_153835 GPR113 G protein-coupled receptor 113 34.24 31.19 Hs.187884 NM_153837 GPR114 G protein-coupled receptor 114 31.86 30.84 Hs.710050 NM_153838 GPR115 G protein-coupled receptor 115 40 23.51 Hs.362806 NM_015234 GPR116 G protein-coupled receptor 116 27.99 21.72 Hs.496762 NM_178471 GPR119 G protein-coupled receptor 119 (*) 35.63 35.98 Hs.123034 NM_005288 GPR12 G protein-coupled receptor 12 33.54 34.47 Hs.435183 NM_001083909 GPR123 G protein-coupled receptor 123 34.4 36.7 Hs.708086 NM_032777 GPR124 G protein-coupled receptor 124 27.98 34.7 Hs.99195 NM_145290 GPR125 G protein-coupled receptor 125 24.46 24.44 Hs.715560 NM_020455 GPR126 G protein-coupled receptor 126 22.45 20.94 Hs.334511 NM_032787 GPR128 G protein-coupled receptor 128 36.17 35.28 Hs.532504 NM_013345 GPR132 G protein-coupled receptor 132 28.86 27.48 Hs.656751 NM_198827 GPR133 G protein-coupled receptor 133 27.94 29.87 Hs.647573 NM_022571 GPR135 G protein-coupled receptor 135 28.69 30.03 Hs.446875 NM_001002911 GPR139 G protein-coupled receptor 139 30.45 29.85 Hs.688230 NM_181791 GPR141 G protein-coupled receptor 141 37.06 39.01 Hs.574368 NM_181790 GPR142 G protein-coupled receptor 142 35.8 34.46 Hs.74124 NM_000273 GPR143 G protein-coupled receptor 143 34.13 27.12 Hs.454099 NM_001161808 GPR144 G protein-coupled receptor 144 33.57 34.42 Hs.729332 NM_138445 GPR146 G protein-coupled receptor 146 31.74 30.75 Hs.452574 NM_207364 GPR148 G protein-coupled receptor 148 35.2 35.27 Hs.688231 NM_001038705 GPR149 G protein-coupled receptor 149 31.07 30.7 Hs.563128 NM_005290 GPR15 G protein-coupled receptor 15 36.47 34.66 Hs.143315 NM_199243 GPR150 G protein-coupled receptor 150 32.57 33.87 Hs.483732 NM_194251 GPR151 G protein-coupled receptor 151 33.16 32 Hs.567997 NM_206997 GPR152 G protein-coupled receptor 152 30.54 30.27 Hs.531581 NM_207370 GPR153 G protein-coupled receptor 153 27.19 27.2 Hs.333358 NM_153002 GPR156 G protein-coupled receptor 156 32.07 31.58 Hs.632367 NM_024980 GPR157 G protein-coupled receptor 157 27.57 25.28 Hs.499108 NM_020752 GPR158 G protein-coupled receptor 158 33.68 31.79 Hs.231320 NM_014373 GPR160 G protein-coupled receptor 160 29.28 24 Hs.271809 NM_153832 GPR161 G protein-coupled receptor 161 25.87 24.7 Hs.631654 NM_014449 GPR162 G protein-coupled receptor 162 26.71 28.97 Hs.46453 NM_005291 GPR17 G protein-coupled receptor 17 31.26 30.45 Hs.549152 NM_013308 GPR171 G protein-coupled receptor 171 33.11 32.54 Hs.661815 NM_018969 GPR173 G protein-coupled receptor 173 28.15 30.92 Hs.326713 NM_032553 GPR174 G protein-coupled receptor 174 38.4 38.98 Hs.37196 NM_007223 GPR176 G protein-coupled receptor 176 21.97 23.55 Hs.462915 NM_001004334 GPR179 G protein-coupled receptor 179 33.88 32.21 Hs.631765 NM_005292 GPR18 G protein-coupled receptor 18 29.78 29.19 Hs.483909 NM_007264 GPR182 G protein-coupled receptor 182 32.29 31.2 Hs.784 NM_004951 GPR183 G protein-coupled receptor 183 27.5 27.52 Hs.657862 NM_006143 GPR19 G protein-coupled receptor 19 31.65 32.27 Hs.188859 NM_005293 GPR20 G protein-coupled receptor 20 34.57 34.59 Hs.728941 NM_005294 GPR21 G protein-coupled receptor 21 31.02 29.77 Hs.657277 NM_005295 GPR22 G protein-coupled receptor 22 31.47 29.99 Hs.534316 NM_005298 GPR25 G protein-coupled receptor 25 37.46 38.18 Hs.12751 NM_153442 GPR26 G protein-coupled receptor 26 (*) 34.83 34.88 Hs.591653 NM_018971 GPR27 G protein-coupled receptor 27 28.07 29.67 Hs.66542 NM_005281 GPR3 G protein-coupled receptor 3 (*) 26.17 26.56 Hs.248124 NM_005299 GPR31 G protein-coupled receptor 31 31 30.4 Hs.515555 NM_001506 GPR32 G protein-coupled receptor 32 36.07 33.68 Hs.495989 NM_005300 GPR34 G protein-coupled receptor 34 31.49 30.5 Hs.239891 NM_005301 GPR35 G protein-coupled receptor 35 34.2 33.59 Hs.406094 NM_005302 GPR37 G protein-coupled receptor 37 24.85 26.61 Hs.132049 NM_004767 GPR37L1 G protein-coupled receptor 37 like 1 32.8 30.17 Hs.432395 NM_001508 GPR39 G protein-coupled receptor 39 28.53 25.46 Hs.17170 NM_005282 GPR4 G protein-coupled receptor 4 29.13 28.77 Hs.299567 NM_004778 PTGDR2 Prostaglandin D2 receptor 2 37.67 37.96 Hs.590903 NM_007227 GPR45 G protein-coupled receptor 45 33.14 35.04 Hs.567390 NM_004224 GPR50 G protein-coupled receptor 50 34.9 34.67 Hs.673850 NM_005684 GPR52 G protein-coupled receptor 52 31.7 31.63 Hs.114545 NM_005683 GPR55 G protein-coupled receptor 55 33.55 33.22 Hs.513633 NM_005682 GPR56 G protein-coupled receptor 56 26.56 24.79 Hs.46332 NM_005284 GPR6 G protein-coupled receptor 6 36.18 37.9 Hs.709782 NM_031936 GPR61 G protein-coupled receptor 61 (*) 37.71 35.49 Hs.232213 NM_080865 GPR62 G protein-coupled receptor 62 35.19 35.44 Hs.632612 NM_030784 GPR63 G protein-coupled receptor 63 28.62 28.31 Hs.146978 NM_005756 GPR64 G protein-coupled receptor 64 30.16 29.06 Hs.513440 NM_003608 GPR65 G protein-coupled receptor 65 (*) 32.03 32.56 Hs.8882 NM_003485 GPR68 G protein-coupled receptor 68 25.81 30.14 Hs.696596 NM_006794 GPR75 G protein-coupled receptor 75 25.52 26.2 Hs.534412 NM_018485 GPR77 G protein-coupled receptor 77 36.11 34.31 Hs.350588 NM_080819 GPR78 G protein-coupled receptor 78 (*) 32.78 32.56 Hs.664795 NM_080817 GPR82 G protein-coupled receptor 82 31.72 30.15 Hs.272385 NM_016540 GPR83 G protein-coupled receptor 83 30.91 30.45 Hs.306199 NM_020370 GPR84 G protein-coupled receptor 84 35.03 33.96 Hs.152009 NM_018970 GPR85 G protein-coupled receptor 85 27.77 31.55 Hs.591292 NM_023915 GPR87 G protein-coupled receptor 87 32.93 25.64 Hs.170053 NM_022049 GPR88 G protein-coupled receptor 88 30.94 30.86 Hs.383403 NM_170776 GPR97 G protein-coupled receptor 97 32.72 32 Hs.591777 NM_032119 GPR98 G protein-coupled receptor 98 35.7 28.62 Hs.631733 NM_003979 GPRC5A G protein-coupled receptor, family C, 23.58 17.06 group 5, member A Hs.148685 NM_016235 GPRC5B G protein-coupled receptor, family C, 29.27 22.56 group 5, member B Hs.446438 NM_018653 GPRC5C G protein-coupled receptor, family C, 33.92 25.51 group 5, member C Hs.644599 NM_018654 GPRC5D G protein-coupled receptor, family C, 29.49 25.92 group 5, member D Hs.266745 NM_148963 GPRC6A G protein-coupled receptor, family C, 34.22 39.42 group 6, member A Hs.128848 NM_000831 GRIK3 Glutamate receptor, ionotropic, kainate 3 40 40 Hs.32945 NM_000838 GRM1 Glutamate receptor, metabotropic 1 36.87 34.53 Hs.121510 NM_000839 GRM2 Glutamate receptor, metabotropic 2 32.26 32.67 Hs.590575 NM_000840 GRM3 Glutamate receptor, metabotropic 3 36.87 32.92 Hs.654847 NM_000841 GRM4 Glutamate receptor, metabotropic 4 33.12 32.9 Hs.147361 NM_000842 GRM5 Glutamate receptor, metabotropic 5 31.84 30.8 Hs.248131 NM_000843 GRM6 Glutamate receptor, metabotropic 6 35.33 33.24 Hs.606393 NM_000844 GRM7 Glutamate receptor, metabotropic 7 33.68 32.69 Hs.449625 NM_000845 GRM8 Glutamate receptor, metabotropic 8 34.05 40 Hs.567282 NM_005314 GRPR Gastrin-releasing peptide receptor 28.1 30.68 Hs.610873 NM_032554 HCAR1 Hydroxycarboxylic acid receptor 1 31.72 30.02 Hs.524812 NM_177551 HCAR2 Hydroxycarboxylic acid receptor 2 33.92 32.17 Hs.388226 NM_001525 HCRTR1 Hypocretin (orexin) receptor 1 33.89 33.48 Hs.151624 NM_001526 HCRTR2 Hypocretin (orexin) receptor 2 37.99 35.2 Hs.1570 NM_000861 HRH1 Histamine receptor H1 25.73 28.32 Hs.247885 NM_022304 HRH2 Histamine receptor H2 32.13 31.68 Hs.251399 NM_007232 HRH3 Histamine receptor H3 31.47 31.07 Hs.287388 NM_021624 HRH4 Histamine receptor H4 33.14 31.81 Hs.247940 NM_000524 HTR1A 5-hydroxytryptamine (serotonin) 35.62 40 receptor 1A Hs.123016 NM_000863 HTR1B 5-hydroxytryptamine (serotonin) 29.48 28.24 receptor 1B Hs.121482 NM_000864 HTR1D 5-hydroxytryptamine (serotonin) 33.86 30.01 receptor 1D Hs.1611 NM_000865 HTR1E 5-hydroxytryptamine (serotonin) 40 37.33 receptor 1E Hs.248136 NM_000866 HTR1F 5-hydroxytryptamine (serotonin) 33.26 35.72 receptor 1F Hs.654586 NM_000621 HTR2A 5-hydroxytryptamine (serotonin) 32.74 33.32 receptor 2A Hs.421649 NM_000867 HTR2B 5-hydroxytryptamine (serotonin) 29.09 28.81 receptor 2B Hs.149037 NM_000868 HTR2C 5-hydroxytryptamine (serotonin) 36.23 40 receptor 2C Hs.413899 NM_000869 HTR3A 5-hydroxytryptamine (serotonin) 33.43 31.46 receptor 3A Hs.241377 NM_006028 HTR3B 5-hydroxytryptamine (serotonin) 33.91 33.52 receptor 3B Hs.483773 NM_000870 HTR4 5-hydroxytryptamine (serotonin) 34.83 34.95 receptor 4 Hs.65791 NM_024012 HTR5A 5-hydroxytryptamine (serotonin) 35.84 34.13 receptor 5A Hs.22180 NM_000871 HTR6 5-hydroxytryptamine (serotonin) 35.5 36.22 receptor 6 Hs.73739 NM_000872 HTR7 5-hydroxytryptamine (serotonin) receptor 7 27.89 30.19 (adenylate cyclase-coupled) (*) Hs.208229 NM_032551 KISSIR KISS1 receptor 34.04 32.88 Hs.705413 NM_002303 LEPR Leptin receptor 26.57 24.8 Hs.502176 NM_018490 LGR4 Leucine-rich repeat containing G 24.47 28.09 protein-coupled receptor 4 Hs.658889 NM_003667 LGR5 Leucine-rich repeat containing G 33.8 33.2 protein-coupled receptor 5 Hs.468490 NM_000233 LHCGR Luteinizing hormone/choriogonadotropin 35.45 34.77 receptor Hs.126667 NM_057159 LPAR1 Lysophosphatidic acid receptor 1 22.02 23.12 Hs.122575 NM_004720 LPAR2 Lysophosphatidic acid receptor 2 29.18 25.33 Hs.674915 NM_012152 LPAR3 Lysophosphatidic acid receptor 3 27.34 29.24 Hs.522701 NM_005296 LPAR4 Lysophosphatidic acid receptor 4 31.29 35.52 Hs.155538 NM_020400 LPAR5 Lysophosphatidic acid receptor 5 33.23 31.2 Hs.123464 NM_005767 LPAR6 Lysophosphatidic acid receptor 6 28.73 26.09 Hs.654658 NM_014921 LPHN1 Latrophilin 1 33.57 29.74 Hs.24212 NM_012302 LPHN2 Latrophilin 2 23.44 22.42 Hs.28391 NM_015236 LPHN3 Latrophilin 3 34.52 28.43 Hs.655431 NM_181657 LTB4R Leukotriene B4 receptor 32.26 31.3 Hs.130685 NM_019839 LTB4R2 Leukotriene B4 receptor 2 31.26 29.47 Hs.99900 NM_002377 MAS1 MAS1 oncogene 36 38.64 Hs.513829 NM_002386 MC1R Melanocortin 1 receptor (alpha melanocyte 30.35 29.34 stimulating hormone receptor) (*) Hs.248144 NM_000529 MC2R Melanocortin 2 receptor 40 40 (adrenocorticotropic hormone) (*) Hs.248018 NM_019888 MC3R Melanocortin 3 receptor (*) 30.98 34.42 Hs.532833 NM_005912 MC4R Melanocortin 4 receptor (*) 35.44 34.64 Hs.248145 NM_005913 MC5R Melanocortin 5 receptor (*) 31.57 31.76 Hs.248122 NM_005297 MCHR1 Melanin-concentrating hormone 34 32.94 receptor 1 Hs.591342 NM_032503 MCHR2 Melanin-concentrating hormone 37.38 36.01 receptor 2 Hs.527802 NM_198923 MRGPRD MAS-related GPR, member D 31.31 30.07 Hs.706565 NM_001039165 MRGPRE MAS-related GPR, member E 31.61 31.18 Hs.118513 NM_145015 MRGPRF MAS-related GPR, member F 25.45 31.62 Hs.730306 NM_001164377 MRGPRG MAS-related GPR, member G 27.3 27.83 Hs.711459 NM_147199 MRGPRX1 MAS-related GPR, member X1 37.05 38.5 Hs.350566 NM_054030 MRGPRX2 MAS-related GPR, member X2 25.94 30.83 Hs.380177 NM_054031 MRGPRX3 MAS-related GPR, member X3 34.05 40 Hs.632138 NM_054032 MRGPRX4 MAS-related GPR, member X4 35.65 35.67 Hs.243467 NM_005958 MTNR1A Melatonin receptor 1A 34.82 33.03 Hs.569039 NM_005959 MTNR1B Melatonin receptor 1B 30.7 30.61 Hs.654478 NM_002511 NMBR Neuromedin B receptor 32.99 34.44 Hs.471619 NM_006056 NMUR1 Neuromedin U receptor 1 33.93 32.54 Hs.283093 NM_020167 NMUR2 Neuromedin U receptor 2 34.63 34.07 Hs.248117 NM_005285 NPBWR1 Neuropeptides B/W receptor 1 36.11 34.44 Hs.248118 NM_005286 NPBWR2 Neuropeptides B/W receptor 2 37.46 36.74 Hs.302026 NM_022146 NPFFR1 Neuropeptide FF receptor 1 34.41 34.73 Hs.99231 NM_053036 NPFFR2 Neuropeptide FF receptor 2 40 29.59 Hs.490330 NM_000906 NPR1 Natriuretic peptide receptor A 28.76 28.04 Hs.78518 NM_003995 NPR2 Natriuretic peptide receptor B 25.45 25.75 Hs.237028 NM_000908 NPR3 Natriuretic peptide receptor B 26.24 27.09 Hs.652373 NM_207172 NPSR1 Neuropeptide S receptor 1 34.05 34.78 Hs.519057 NM_000909 NPY1R Neuropeptide Y receptor Y1 32.34 30.81 Hs.37125 NM_000910 NPY2R Neuropeptide Y receptor Y2 30.61 29.84 Hs.598503 NM_006174 NPY5R Neuropeptide Y receptor Y5 38.24 35.21 Hs.590869 NM_002531 NTSR1 Neurotensin receptor 1 (high affinity) 27.66 32.7 Hs.131138 NM_012344 NTSR2 Neurotensin receptor 2 34.14 33.3 Hs.677835 NM_181745 O3FAR1 Omega-3 fatty acid receptor 1 35.31 32.92 Hs.67896 NM_007346 OGFR Opioid growth factor receptor 26.18 25.76 Hs.656404 NM_001708 OPN1SW Opsin 1 (cone pigments), 24.49 24.73 short-wave-sensitive Hs.534399 NM_014322 OPN3 Opsin 3 26.3 24.9 Hs.283922 NM_033282 OPN4 Opsin 4 33.61 33.56 Hs.213717 NM_181744 OPN5 Opsin 5 40 32.7 Hs.372 NM_000911 OPRD1 Opioid receptor, delta 1 30.86 29.97 Hs.106795 NM_000912 OPRK1 Opioid receptor, kappa 1 34.64 34.46 Hs.2859 NM_000913 OPRL1 Opiate receptor-like 1 33.94 31.44 Hs.2353 NM_000914 OPRM1 Opioid receptor, mu 1 35.48 37.77 Hs.352218 NM_080818 OXGR1 Oxoglutarate (alpha-ketoglutarate) 40 37.8 receptor 1 Hs.2820 NM_000916 OXTR Oxytocin receptor 25.26 25.31 Hs.654526 NM_002563 P2RY1 Purinergic receptor P2Y, G-protein 29.28 29.49 coupled, 1 Hs.296433 NM_198333 P2RY10 Purinergic receptor P2Y, G-protein 40 40 coupled, 10 Hs.166168 NM_002566 P2RY11 Purinergic receptor P2Y, G-protein 31.61 31.58 coupled, 11 Hs.591281 NM_022788 P2RY12 Purinergic receptor P2Y, G-protein 32.42 32.59 coupled, 12 Hs.546396 NM_176894 P2RY13 Purinergic receptor P2Y, G-protein 33.65 33.98 coupled, 13 Hs.2465 NM_014879 P2RY14 Purinergic receptor P2Y, G-protein 34.63 34.32 coupled, 14 Hs.339 NM_002564 P2RY2 Purinergic receptor P2Y, G-protein 35.47 27.48 coupled, 2 Hs.673854 NM_002565 P2RY4 Pyrimidinergic receptor P2Y, 34.14 32.57 G-protein coupled, 4 Hs.16362 NM_004154 P2RY6 Pyrimidinergic receptor P2Y, 34.56 31.31 G-protein coupled, 6 Hs.111377 NM_178129 P2RY8 Purinergic receptor P2Y, G-protein 36.9 40 coupled, 8 Hs.509067 NM_002609 PDGFRB Platelet-derived growth factor 27.58 32.28 receptor, beta polypeptide Hs.458573 NM_006207 PDGFRL Platelet-derived growth factor 26.52 27.21 receptor-like Hs.524719 NM_005972 PPYR1 Pancreatic polypeptide receptor 1 29 28.22 Hs.248119 NM_004248 PRLHR Prolactin releasing hormone receptor 32.86 32.31 Hs.683430 NM_138964 PROKR1 Prokineticin receptor 1 40 35.42 Hs.375029 NM_144773 PROKR2 Prokineticin receptor 2 35.49 34.73 Hs.709174 NM_000952 PTAFR Platelet-activating factor receptor 31.98 29.55 Hs.306831 NM_000953 PTGDR Prostaglandin D2 receptor (DP) (*) 25.3 24.34 Hs.159360 NM_000955 PTGER1 Prostaglandin E receptor 1 32.17 29.07 (subtype EP1), 42 kDa Hs.2090 NM_000956 PTGER2 Prostaglandin E receptor 2 25.8 25.86 (subtype EP2), 53 kDa (*) Hs.445000 NM_198715 PTGER3 Prostaglandin E receptor 3 (subtype EP3) 26.95 29.88 Hs.199248 NM_000958 PTGER4 Prostaglandin E receptor 4 (subtype EP4) 26.62 25.16 Hs.654365 NM_000959 PTGFR Prostaglandin F receptor (FP) 27.13 33.28 Hs.458324 NM_000960 PTGIR Prostaglandin I2 (prostacyclin) 28.32 30.43 receptor (IP) Hs.1019 NM_000316 PTH1R Parathyroid hormone 1 receptor 33.51 32.47 Hs.570296 NM_005048 PTH2R Parathyroid hormone 2 receptor 36.11 36.96 Hs.368977 NM_198179 QRFPR Pyroglutamylated RFamide peptide receptor 34.89 40 Hs.1544 NM_002921 RGR Retinal G protein coupled receptor 33.43 33.89 Hs.247565 NM_000539 RHO Rhodopsin 31.99 32.3 Hs.658310 NM_006583 RRH Retinal pigment epithelium-derived 33.04 32.26 rhodopsin homolog Hs.591686 NM_021634 RXFP1 Relaxin/insulin-like family peptide 33.24 36.53 receptor 1 Hs.680763 NM_130806 RXFP2 Relaxin/insulin-like family peptide 33.27 32.82 receptor 2 Hs.170146 NM_016568 RXFP3 Relaxin/insulin-like family peptide 33.21 33.03 receptor 3 Hs.449914 NM_181885 RXFP4 Relaxin/insulin-like family peptide 34 32.75 receptor 4 Hs.154210 NM_001400 S1PR1 Sphingosine-1-phosphate receptor 1 26.42 28.45 Hs.655405 NM_004230 S1PR2 Sphingosine-1-phosphate receptor 2 23.97 25.2 Hs.585118 NM_005226 S1PR3 Sphingosine-1-phosphate receptor 3 25.59 26.58 Hs.662006 NM_003775 S1PR4 Sphingosine-1-phosphate receptor 4 33.52 30.76 Hs.501561 NM_030760 S1PR5 Sphingosine-1-phosphate receptor 5 37.46 36.16 Hs.42091 NM_002980 SCTR Secretin receptor 40 40 Hs.522087 NM_005866 SIGMAR1 Sigma non-opioid intracellular 21.57 22.06 receptor 1 Hs.437846 NM_005631 SMO Smoothened, frizzled family receptor 27.8 30.67 Hs.591915 NM_052918 SORCS1 Sortilin-related VPS10 domain containing 32.14 33.05 receptor 1 Hs.479099 NM_020777 SORCS2 Sortilin-related VPS10 domain containing 32.76 32.61 receptor 2 Hs.671950 NM_014978 SORCS3 Sortilin-related VPS10 domain containing 40 30.11 receptor 3 Hs.248160 NM_001049 SSTR1 Somatostatin receptor 1 23.91 29.84 Hs.514451 NM_001050 SSTR2 Somatostatin receptor 2 32.93 32.08 Hs.225995 NM_001051 SSTR3 Somatostatin receptor 3 33.57 33.23 Hs.673846 NM_001052 SSTR4 Somatostatin receptor 4 31.45 31.61 Hs.449840 NM_001053 SSTR5 Somatostatin receptor 5 40 37.35 Hs.279575 NM_033050 SUCNR1 Succinate receptor 1 34.48 34.88 Hs.375030 NM_138327 TAAR1 Trace amine associated receptor 1 35.07 34.03 Hs.272382 NM_014626 TAAR2 Trace amine associated receptor 2 36.98 40 Hs.248198 NM_003967 TAAR5 Trace amine associated receptor 5 27.07 30.8 Hs.434196 NM_175067 TAAR6 Trace amine associated receptor 6 33.81 33.85 Hs.434116 NM_175057 TAAR9 Trace amine associated receptor 9 35.81 40 (gene/pseudogene) Hs.633301 NM_001058 TACR1 Tachykinin receptor 1 28.18 31.06 Hs.88372 NM_001057 TACR2 Tachykinin receptor 2 28.21 29.21 Hs.942 NM_001059 TACR3 Tachykinin receptor 3 31.34 31.62 Hs.442530 NM_001060 TBXA2R Thromboxane A2 receptor 29.62 31.6 Hs.656790 NM_032027 TM2D1 TM2 domain containing 1 24.13 23.97 Hs.3022 NM_003301 TRHR Thyrotropin-releasing hormone receptor 32.54 32.8 Hs.160411 NM_000369 TSHR Thyroid stimulating hormone receptor 34.52 35.82 Hs.192720 NM_018949 UTS2R Urotensin 2 receptor 32.55 31.77 Hs.348500 NM_004624 VIPR1 Vasoactive intestinal peptide receptor 1 34.58 26.91 Hs.585052 NM_003382 VIPR2 Vasoactive intestinal peptide receptor 2 (*) 35.05 33.28 Hs.248116 NM_005283 XCR1 Chemokine (C motif) receptor 1 35.03 32.98 Hs.227656 NM_004736 XPR1 Xenotropic and polytropic retrovirus 23.47 22.49 receptor 1 Hs.592355 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 17.39 17.41

For all other in vitro experiments, cells were treated as indicated prior to RNA isolation using RNeasy Plus Mini Kit (Qiagen) according to manufacturer's instructions. Isolated RNA (250 ng) was then used to synthesize cDNA using SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using a LightCycler 96 (Roche). Data are expressed as a fold change by ΔΔCt relative to GAPDH. For in vivo experiments, tissue was immediately frozen, and stored at −80° C. RNA isolation, cDNA synthesis, and qPCR analysis were performed as above. Primers used for qPCR are shown in Table 2.

TABLE 2 Human Primer Sequence DRD 1 F: CCCAGCCCTATCAGTCATATTG,  SEQ ID NOs:  R: AGGATTCATCTGCGAGTTCAG 1 and 2 CTGF F: GTCCAGCACGAGGCTCA,  SEQ ID NOs:  R: TCGCCTTCGTGGTCCTC 3 and 4 COL1A1 F: AAGGGACACAGAGGTTTCAGTGG,  SEQ ID NOs:  R: CAGCACCAGTAGCACCATCATTTC 5 and 6 ACTA2 F: GTGAAGAAGAGGACAGCACTG,  SEQ ID NOs:  R: CCCATTCCCACCATC ACC 7 and 8 FN1 F: TGTCAGTCAAAGCAAGCCCG,  SEQ ID NOs:  R: TTAGGACGCTCATAAGTGTCACCC 9 and 10 TGM2 F: TCAGCTACAATGGGATCTTGG,  SEQ ID NOs:  R: AAGGCAGTCACGGTATTTCTC 11 and 12 LOX F: ACATTCGCTACACAGGACATC,  SEQ ID NOs:  R: TTCCCACTTCAGAACACCAG 13 and 14 LOXL1 F: TGCCAGTGGATCGACATAAC,  SEQ ID NOs:  R: GAAACGTAGCGACCTGTGTAG 15 and 16 LOXL2 F: GTGCAGCGACAAAAGGATTC,  SEQ ID NOs:  R: GCGGTAGGTTGAGAGGATG 17 and 18 LOXL3 F: AGCGAAAAGAGGGTCAACG,  SEQ ID NOs:  R: TGTCATTGGCACGATAGAACTC 19 and 20 LOXL4 F: GTGGCAGAGTCAGATTTCTCC,  SEQ ID NOs:  R: TTGTTCCTGAGACGCTGTTC 21 and 22 PLAU F: GGGAGATGAAGTTTGAGGTGG,  SEQ ID NOs:  R: AGATGGTCTGTATAGTCCGGG 23 and 24 PLAT F: AAACCCAGATCGAGACTCAAAG,  SEQ ID NOs:  R: ACCCATTCCCAAAGTAGCAG 25 and 26 CTSK F: CTCCTTCCAGTTTTACAGCAAAG,  SEQ ID NOs:  R: TTTCCCCAGTTTTCTCCCC 27 and 28 MMP14 F: TGCCTACCGACAAGATTGATG,  SEQ ID NOs:  R: ATCCCTTCCCAGACTTTGATG 29 and 30 Mouse Primer Sequence Drd1 F: CCCGTAGCCATTATGATCGTC,  SEQ ID NOs:  R: AGAGCATTCGACAGGGTTTC 31 and 32 Pdgfra F: TCCTTCTACCACCTCAGCGAG,  SEQ ID NOs:  R: CCGGATGGTCACTCTTTAGGAAG 33 and 34 Epcam F: TTGCTCCAAACTGGCGTCTA,  SEQ ID NOs:  R: ACGTGATCTCCGTGTCCTTGT 35 and 36 Pecam 1 F: CTGCCAGTCCGAAAATGGAAC,  SEQ ID NOs:  R: CTTCATCCACTGGGGCTATC 37 and 38 Ptprc F: GACAGAGTTAGTGAATGGAGACC,  SEQ ID NOs:  R: AAAAGTTCGGAGAGTGTAGGC 39 and 40 Acta2 F: GAGAAGCCCAGCCAGTCG,  SEQ ID NOs:  R: CTCTTGCTCTGGGCTTCA 41 and 42 Ctgf F: CCTGCGACCCACACAAG,  SEQ ID NOs:  R: GACCCACCGAAGACACAG 43 and 44 Fnl F: CCAGCAGCATGATCAAAACAC,  SEQ ID NOs:  R: GGTGGCTACATGTTAGAGTGTC 45 and 46 Collal F: ATCATAGCCATAGGACATCTGG,  SEQ ID NOs:  R: CTGGACAGCCTGGACTTC 47 and 48 Yap1 F: CTCTGAGTGATCCTCTGGTTC,  SEQ ID NOs:  R: CCATAAGAACAAGACCACATCCT 49 and 50 WWtr1 F: CTTGCTGGTGTTGGTGATTC,  SEQ ID NOs:  R: ATCAGCCTCTGAATCATGTGAA 51 and 52 Alb F: TGCTTTTTCCAGGGGTGTGTT,  SEQ ID NOs:  R: TTACTTCCTGCACTAATTTGGCA 53 and 54

Cell Sorting: FACS: PBS perfused mouse lungs were finely minced with a razor blade in a 100 mm petri dish in 1 mL of cold DMEM medium containing 0.2 mg/ml Liberase DL (Roche) and 100 U/ml DNase I (Roche). The mixture was transferred to 15 ml tubes and incubated at 37° C. for 30 min in a water bath. Enzymatic digestion was inactivated by adding DMEM medium containing 10% fetal bovine serum. The cell suspension was passed once through a 40 μm cell strainer (Fisher) to remove multicellular debris. Cells were then centrifuged at 1,300 r.p.m. at 4° C. for 10 min, washed once in PBS and resuspended in 0.2 ml of FACS buffer (1% BSA, 0.5 μM EDTA pH 7.4 in PBS). The single cell suspension was then incubated with anti-CD45-PerCp-Cy5.5, anti-CD31-PE, anti-PDGFRα-APC and anti-EpCAM-BV421 antibodies (1:200) (Biolegend) for 20 min on ice. After incubation, cells were washed with ice-cold FACS buffer and resuspended in 1 ml of FACS buffer. FACS sorting was conducted using a BD FACS Aria II (BD Biosciences). FACS-sorted epithelial cells, endothelial cells and fibroblasts were collected in 1.5 mL Eppendorf tubes containing RLT lysis buffer (Qiagen), which were subjected to mRNA extraction, complementary DNA synthesis and RT-PCR analysis. MACS: PBS perfused mouse lungs were finely minced with a razor blade in a 100 mm petri dish in 1 ml of MACs dissociation solution described in the MACS mouse lung dissociation kit. The mixture was transferred to 15 ml tubes and incubated at 37° C. for 30 min in a water bath. Enzymatic digestion was inactivated by adding DMEM containing 10% fetal bovine serum. The cell suspension was passed once through a 40 μm cell strainer (Fisher) to remove multicellular debris. Cells were then centrifuged at 1,350 r.p.m. at 4° C. for 10 min and supernatant aspirated. The samples were resuspended in 0.1 mL 15% BSA-autoMACS rinsing solution. The single cell suspension was than incubated with mouse anti-CD45 MicroBeads (1:10) for 15 minutes at 4-8° C. Cells were then magnetically filtered using LS column (Miltenyi Biotec). Positively selected cells were pelleted at 1350 RPM at 4° C. and resuspended in RLT lysis buffer (Qiagen). Samples were then subjected to mRNA extraction, complementary DNA synthesis and RT-PCR analysis.

Immunofluorescence Microscopy: Cells were plated into 96 well plates (Corning 3603) in their specific growth media and allowed to attach (8 hours). Media was then exchanged for the indicated conditions for each experiment. Cells were fixed in 3.7% formalin (Sigma-Aldrich), permeabilized in 0.25% Triton X-100 (Sigma-Aldrich) and then blocked with 1% BSA for 1 h. Cells or tissue sections were incubated overnight with mouse monoclonal antibody against αSMA (Sigma-Aldrich F3777), and/or a rabbit monoclonal antibody against YAP/TAZ (Cell Signaling D24E4) diluted 1:200 in PBS with 1% BSA. Cells were then washed and exposed to flourescence-conjugated secondary antibodies (Invitrogen) diluted 1:1000 and DAPI (Thermo Fisher Scientific). Images were taken with a Cytation5 (BioTek) microscope. For scoring αSMA positive cells (FIG. 12), an observer blinded to the treatment conditions counted αSMA-positive cells using a visual threshold for bright fibrous staining; a minimum of 200 cells was counted for each condition. YAP/TAZ localization was quantified (FIGS. 6-10, 24-26, and 29-30) using Gen5 (Biotek) software. Images were taken at 4× magnification of both DAPI and YAP/TAZ staining. Objects were identified using the DAPI channel and a subpopulation of YAP/TAZ nuclear positive cells was counted based on nuclei where the average pixel intensity of the YAP/TAZ channel was greater than 85% of the average pixel intensity of all the nuclei in the control treated cells. Quantification of double positive (YAP/TAZ and αSMA) cells from lung tissue sections (FIG. 17-21) was performed similarly; however separate thresholds were established for both YAP/TAZ and αSMA. A minimum of 4000 cells was quantified for each mouse.

Traction Force Microscopy: Traction analysis was conducted as previously described. Briefly, polyacrylamide substrates with shear moduli of 6.4 kPa were prepared, and fluorescent sulfate-modified latex microspheres (0.2 μm, 505/515 ex/em) (FluoSpheres, Life Technologies) were conjugated to the gel surfaces after treatment with 1 mg/ml of dopamine hydrochloride (Sigma-Aldrich) in 50 mM HEPES solution (pH 8.5). IPF patient derived fibroblasts were plated on the gels overnight and treated as indicated before traction force measurements. Images of gel surface-conjugated fluorescent beads were acquired for each cell before and after trypsinization using a Nikon ECLIPSE Ti microscope at ×10 magnification. Traction forces were estimated by measuring bead displacement fields and computing corresponding traction fields using TractionsForAll (freely distributed program that calculates 2-D tractions exerted by an adherent cell on its substrate).

cAMPAssay: IPF patient derived fibroblasts were plated in EMEM containing 10% FBS overnight. Media was exchanged with EMEM containing 0.1% FBS for 24 hours. cAMP was measured using the cAMP-Glo™ Assay (Promega) according to manufacturer's suggestions. 20 minutes prior to cell lysis media was removed and cells were treated with “induction buffer” containing nonselective phosphodiesterase inhibitors and the indicated concentration of compound(s). Luminescence was measured on a Flexstation 3 (Molecular Devices) plate reader.

Western Blotting: Cells were plated in EMEM containing 10% FBS overnight. Media was exchanged with EMEM containing 0.1% FBS for 24 hours. Prior to protein isolation cells were treated with the indicated concentration of compounds for the indicated time. Total protein was isolated using RIPA buffer (pH 8.0) containing Pierce Phosphatase Inhibitor (Thermo) and Halt Protease Inhibitor Cocktail (Thermo). Lysate total protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo) and samples were run on a 10% polyacrylamide gel. Blots were incubated overnight with primary antibodies: pYAP (Ser 127, Cell Signaling D9W21), YAP/TAZ (Cell Signaling D24E4), GAPDH (Cell Signaling 14C10), HSC70 (Santa Cruz sc-7298), αSMA (Abcam ab5694), and fibronectin (Santa Cruz sc-9068) diluted 1:1000 in Li-Cor Odyssey Blocking Buffer. Blots were washed with TBS-Tween before 60 minute incubation with IR-dye-conjugated secondary antibodies (Li-Cor) diluted 1:10,000. Plates were imaged via a Li-Cor OdysseyXL system with quantification performed via densitometry.

Immuno-ECM Adapting from previously published methods, IPF patient-derived fibroblasts were plated to confluence in clear-bottom 96-well plates. After cells attached, the medium was swapped for EMEM containing 0.10% FBS±2 ng/mL TGF-β. After 48 hours the indicated concentration of DHX or DMSO control was added to each well and incubated for 24 hours. WST-1 viability reagent was added to each well (Sigma-Aldrich) and measured on a Flexstation 3 (Molecular Devices) plate reader. Cells were then fixed in 3.7% formalin (Sigma-Aldrich), and permeabilized in 0.25% Triton X-100 (Sigma-Aldrich). Wells were washed with tris-buffered saline (TBS) and blocked with Li-Cor Odyssey Blocking Buffer for 60 minutes before overnight incubation in a polyclonal rabbit antibody for fibronectin (Sigma sc-9068) or collagen I (Novus NB600-408) diluted 1:200 in blocking buffer. Wells were washed with TBS-Tween before 45 minute incubation with IR-dye-conjugated secondary antibody (Li-Cor #926-32211) diluted 1:400. Plates were imaged via a Li-Cor OdysseyXL system with quantification performed via densitometry. Data are expressed as IR intensity relative to WST-1 signal absorbance in order to account for any potential compound toxicity.

Matrix Remodeling Measured by Atomic Force Microscopy: NIH-3T3 cells were plated to confluence onto gelatin coated (Cell Biologics) AFM compatible tissue culture dishes (Willco) in DMEM containing 10% FBS. After cells attached overnight media was replaced with DMEM containing 2% FBS, 2 ng/mL TGFβ, and 20 μg/mL ascorbic acid to promote matrix deposition. After 72 hours, measurements were made using a BioScope Catalyst AFM (Bruker, MA, USA). Microindentations were performed using a 2.5 μm radius sphere-tipped probe (Novascan, IA, USA) with a spring constant determined at about 100 pN/nm by thermal fluctuation method. For each dish, 3 different areas were analyzed. Force curves were acquired with MIRO 2.0 (NanoScope 9.1; Bruker) at an indentation rate of 20 μm/s and a ramp size of 10 μm on different points. 75 force curves were performed per cell dish (25 per area). The Young's modulus E was determined by the fitting of force curve by Hertz model using NanoScope Analysis software (Bruker) and considering Poisson's ratio of 0.5. Media was exchanged for fresh DMEM containing 2% FBS, 2 ng/mL TGFβ, and 20 μg/mL ascorbic acid with the indicated concentration of DHX or 0.1% DMSO (vehicle control). After another 72 hours AFM measurements were made as before. The resulting cell-derived matrix was then decellularized with Phosphate-buffered saline (Gibco) containing: 0.5% (v/v) Triton X-100 (Sigma-Aldrich) and 20 mM NH₄OH (LabChem Inc.). Matrices were washed 3× with PBS and then plated with low passage (P3), NHLFs for 24 hours prior to RNA isolation.

RNA Interference: Cells were transfected using Lipofectamine RNAiMAX (Life Technologies) with 25 nM siGENOME siRNA SMARTpool (Dharmacon) targeting DRD1 (L-005477-00-0005) or a nontargeting SMARTpool (D-001810-10-05). Cells were cultured for 72 hours before collecting RNA. For the YAP/TAZ localization experiments, the cells were transfected in their 6-well plates for 48 hours prior to re-plating into 96-well plates for the immunofluorescence assays. See below for in vivo siRNA methodology.

Bleomycin Mouse Study: In the initial in vivo siRNA study (FIGS. 22 and 23A-23C), adult male age-matched C57BL/6N mice at 6-8 weeks of age were purchased from the National Cancer Institute (NCI)-Frederick Mouse Repository (Frederick, Md., USA). All experiments were performed in accordance with National Institute of Health guidelines and protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care, and maintained all mice in a specific pathogen-free (SPF) environment certified by the American Association for Accreditation of Laboratory Animal Care (AAALAC). 6-8 weeks old mice were anesthetized with ketamine and xylazine before exposure of the trachea. Lung fibrosis was induced by intratracheal injection of bleomycin (50 μl at 1.2 U/kg) or phosphate buffered saline (PBS; as control) on day 0. After 14 days Small interfering RNA (siRNA) duplexes targeting mouse Yap (L-046247-01-0005) or Taz (L-058248-01-0005) mRNA (Dharmacon) or nontargeting control siRNA were administered in vivo by intratracheal instillation at a single dose of 25 μg (each siRNA) per mouse. On day 21 Mice were sacrificed and lungs harvested for collagen determination and biochemical analyses. To obtain BAL samples for total protein concentration determination, lungs were lavaged with six 0.5-mL aliquots of PBS. BAL samples were centrifuged at 3,000 g for 20 min at 4° C. and transferred the supernatants to siliconized low-binding Eppendorf tubes (PGC Scientifics) for subsequent analysis. Total protein concentration of the BAL fluid was determined by BCA Protein Assay Kit (Pierce). In the dihydrexidine treatment studies (FIG. 17-21), 8 week old female C57/BL6 mice were purchased from Charles River Laboratories. Mouse lung fibrosis was induced with bleomycin (BLEO; Fresenius Kabi) delivered intratracheally (3 U/kg) to the lungs using MicroSprayer® Aerosolizer (Penn-Century). The Sham mice received sterile 0.9% saline instead using identical methods. Mice were weighed every 24 hrs, and both groups were then randomized at day 10 into DHX and Control treatment groups, matching for the degree of weight change. DHX (5 mg/kg) was administered everyday intranasally (i.n.) dissolved in surfactant (infasurf) which has previously shown to aid in spreading to pulmonary aveoli for 14 days. The control groups of mice received the equivalent vehicle dose of surfactant. Following the final DHX treatment, mice were sacrificed and the right lungs were inflated with 4% paraformaldehyde (PFA) and further incubated in 4% PFA for 24 hours prior processing for paraffin embedding. The left lobe of the lung was snap frozen in liquid nitrogen for RNA isolation and hydroxyproline assay. Experimental procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee and the animals were handled in accordance with their guidelines.

Bile Duct Ligation: BDL was performed as previously described. Briefly, 8-10 weak old female C57BL/6N underwent either BDL or sham surgery. Mice were anesthetized on Day 0 following IACUC protocol, and the bile duct was ligated using sterile 3/0 silk ligatures. Sham surgery was performed by passing a silk ligature under the bile duct. Starting on Day 7, DHX (5 mg/kg) or vehicle control was administered everyday intraperitoneally (i.p.) for 14 days. Following the final DHX treatment, mice were sacrificed and the livers harvested for analysis of fibrosis.

Histological Scoring: Five μm thick sections were cut from Paraffin embedded lung tissues, and the sections were stained either with hematoxylin and eosin (H&E) or with Masson's Trichrome stain kit (Abcam). All H&E-stained slides and trichrome-stained slides were reviewed in a blinded fashion by a thoracic pathologist. The severity of interstitial and peribronchiolar lung immature and mature fibrosis was estimated on a numerical scale according to Ashcroft et al. For scoring purposes, all H&E stained slides were systematically scanned at 100× magnification and successive 100× fields were scored. Scoring was based on the following scale: 0 (no fibrosis), 1 (minimal interstitial and/or peribronchiolar thickening due to fibrosis), 3 (moderate thickening without obvious architectural distortion), 5 (increased fibrosis with formation of fibrous bands and/or small masses), 7 (severe architectural distortion with large areas of fibrosis and areas of honeycomb changes), and 8 (total fibrous obliteration of the field). The predominant score for each field was recorded. The mean of all scores was calculated for each case. Liver trichrome stained sections were computationally measured using Image J software. After converting each image in an RGB stack, the threshold was adjusted and kept at the same level for all the images.

Hydroxyproline: Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision) according to the manufacture's instruction with slight modification. The lung tissues were weighed, homogenized in sterile water (10 mg of tissue per 100 μL H₂O) and hydrolyzed in 12N HCl in a pressure-tight, teflon capped vial at 120° C. for 3 hours followed by filtration through a 45 μm Spin-X® Centrifuge Tube filter (Corning). 10 μL of the hydrolyzed samples was dried in a Speed-Vac for 2 hours, followed by incubation with 100 μL of Chloramine T reagent for 5 minutes at room temperature and 100 μL of 4□(dimethylamino) benzaldehyde (DMAB) for 90 minutes at 60° C. The absorbance of oxidized hydroxyproline was determined at 560 nm. Hyrdroxyproline concentrations were calculated from a standard curve generated using known concentrations of trans-4-hydroxyl-L-proline. The total amount of protein isolated from the weighed tissues was determined by using a protein assay kit (Bio-Rad, absorbance at 595 nm). The amount of collagen was expressed in μg/mg total protein.

Statistics: Groups were compared by one-way ANOVA with Tukey's multiple comparison's test. All statistical tests were carried out using GraphPad Prism 6 with statistical significance defined asp <0.05. Results are expressed throughout as the mean±standard error of the mean (SEM).

Example 1—Selective YAP and TAZ Targeting by Agonizing Gα_(s) Receptors

To test whether nonselective YAP and TAZ targeting may be effective in a model of pulmonary fibrosis, YAP and TAZ siRNA were administered intratracheally to mice following bleomycin injury, a standard model of pulmonary fibrosis (FIG. 17-21). Non-selective targeting of YAP/TAZ in this context amplified fibrosis (measured by hydroxyproline assay), while also increasing lung injury and vascular leakage. Contrastingly, fibroblast selective genetic deletion of YAP and TAZ has shown promise in a kidney fibrosis model (See e.g., Ref. 25).

G protein-coupled receptors (GPCRs) make up the largest family of membrane receptors in the human genome, and have been prolific therapeutic targets, with their ligands account for >30% of all clinically approved drugs (See e.g., Ref. 26). GPCRs are linked to effector proteins from four main classes of G-proteins. Activation of receptors which couple to Gα_(12/13), Gα_(q/11) and Gα_(i/o) stimulates YAP/TAZ nuclear translocation and transcriptional activity. In contrast, receptors which couple to Gα_(s) inhibit YAP/TAZ nuclear localization and activity via elevation of cAMP (See e.g., Ref. 27-30) (FIG. 1,2).

GPCR expression varies across organs and even within adjacent cell types in the same tissue (See, e.g., Ref. 31). Therefore, RNA expression of the GPCRome was profiled in both primary adult human pulmonary fibroblasts and alveolar epithelial cells (FIG. 3), searching for receptors which exclusively couple to Gα_(s) (See, e.g., Ref. 32) (larger dots, FIG. 3) and are expressed selectively in fibroblasts. Of the 28 Gα_(s) coupled receptors, expression of the Dopamine Receptor D1 (DRD1) exhibited relatively high expression and pronounced enrichment in fibroblasts compared to alveolar epithelial cells (FIG. 3). Abundant transcripts for DRD1 in cultured normal human lung fibroblasts and fibroblasts derived from patients with idiopathic pulmonary fibrosis was determined by qPCR, and undetectable transcript levels of DRD1 in both primary human alveolar epithelial and microvasculature endothelial cells (FIG. 4). To further validate our findings in freshly isolated lung cell populations, we sorted mouse lung tissue into mesenchymal, epithelial, endothelial, and leukocyte enriched fractions (FIG. 5). As in cultured human cells we observed robust expression of DRD1 in the freshly isolated mesenchymal cells, but undetectable levels in other lung cell populations.

Example 2—DRD1 Agonists Selectively Inhibit YAP and TAZ Localization in Mesenchymal Cells

Three selective DRD1 agonists (dihydrexidine, SKF-81297, fenoldopam) were tested for their ability to inhibit YAP/TAZ nuclear localization (FIG. 6, 24). Fibroblasts plated on stiff matrix (plastic) and lacking cell contact inhibition have abundant nuclear localization of YAP/TAZ (See e.g., Ref. 2). All three compounds reduced nuclear localization of YAP/TAZ, and their efficacy was consistent with previously described intrinsic activity of these ligands (See e.g., Ref. 33).

The inhibition of YAP/TAZ nuclear localization by dihydrexidine (DHX) could be attenuated using a DRD1 selective antagonist (FIG. 24-26) or by treating the cells with DRD1-siRNA (FIG. 27-28), confirming the receptor-specific effects of DHX. Consistent with the previously defined mechanism whereby YAP/TAZ nuclear localization is controlled by cAMP-dependent phosphorylation of serine residues, promoting cytoplasmic retention or degradation, DHX elevated cAMP and promoted YAP serine 127 phosphorylation (See e.g., Ref. 27-30) (FIG. 9, 10). DHX was effective at inhibiting YAP/TAZ nuclear localization across a panel of mesenchymal cell types, including cardiac and dermal fibroblasts and hepatic stellate cells (FIG. 24-26), suggesting potentially broad relevance of this ligand for mesenchymal cell targeting of YAP and TAZ. DHX-mediated inhibition of YAP/TAZ nuclear localization was relatively sustained, and equally potent in normal lung fibroblasts and those derived from a patient with IPF, unlike the reduced potency of another GPCR ligand (PGE₂) with known anti-fibrotic effects in the lung (FIG. 24-26) (See e.g., Ref. 34). In contrast to these observations, DHX had no effect on YAP/TAZ localization in pulmonary epithelial and endothelial cells (FIG. 7), consistent with the absence of detectable transcripts for DRD1 in these cell types. Multiple GPCR ligands which are known to promote fibrosis couple to Gα_(i), Gα_(q), and Gα₁₂ which would in turn be expected to enhance YAP/TAZ nuclear localization in fibroblasts (See e.g., Ref. 28, 33). It was confirmed in confluent fibroblasts, which otherwise exhibit reduced nuclear localization of YAP/TAZ, showing that endothelin-1, lysophosphatidic acid and serotonin, all GPCR ligands implicated in promotion of fibrosis (See e.g., Ref. 35), enhance YAP/TAZ nuclear localization. DHX blocked nuclear localization of YAP/TAZ in responses to all three of these ligands, demonstrating the broad effects of GPCR ligands on YAP/TAZ in fibroblasts, and identifying DHX and stimulation of Gα_(s)/cAMP as an effective strategy for inhibiting both mechanical (stiff matrix) and biochemical regulation of YAP/TAZ nuclear localization (FIG. 8). The ability of selected dopamine receptor agonists to inhibit YAP/TAZ nuclear localization is shown in Table 3.

TABLE 3 Nuclear localization (% of total cells) Compound IPF-1 IPF-2 IPF-3 IPF-4 DMSO 89.96719 77.08034 76.3871 73.26695 R(−)-2,10,11-Trihydroxyaporphine•HBr 13.70637 22.14634 24.15385 17.75309 Dihydrexidine 13.22484 21.79444 27.63449 21.08058 A 68930•HCl 33.12535 30.59259 26.82353 10.25995 (R)-(−)-Apomorphine•HCl 29.20735 31.96825 27.47368 17.86425 (±)-SKF-82958•HBr 29.15385 38 38 12.28571 CY 208-243 20.51748 39.79487 41.17073 19.81818 R(−)-Propylnorapomorphine•HCl 28.34884 26.70968 42.73684 29.03004 R(+)-6-BROMO-APB•HBr 28.36697 38 36 25.64706 R(−)-2,10,11-Trihydroxy-N-propyl- 32.86874 35.25275 37.69697 25.35849 noraporphine•HBr A-77636•HCl•H₂O 26.46154 30.88136 41.33333 37.78903 Dopamine•HCl 39.84783 59.28571 49.66667 49.50943 6,7-ADTN•HBr 43.88865 44 65.5 52.64912 Mesulergine•HCl 37.81413 45.14286 78.625 45.14286 SKF 38393•HBr 69.5873 47.375 61.33333 35.79116 N-Methyldopamine•HCl 43.15829 46.73016 60.10526 70.44275 4-Hydroxyphenethylamine•HCl 56.51852 45.69231 76.57143 43.3719 Cabergoline 57.01515 57.41176 45.95181 68.69498 3-Hydroxyphenethylamine•HCl 48.95506 56.91892 69.25 58.37466 Pramipexole Dihydrochloride Monohydrate 64.92308 47.64912 65.5 58.96774 PD 168077 maleate 67.07869 42.44444 63.67568 68.4 Fenoldopam•HCl 68.09828 50.31884 66.63636 63 (±)-PD 128,907•HCl 60.13115 49.53846 78.47619 60.66187 (±)-2-(N-phenylethyl-N-propyl)amino-5- 68.85 60.85714 68 56.552 hydroxytetralin•HCl Bromocriptine mesylate 73.02879 52.81481 64.08696 64.59574 Ropinirole HCl 69.6036 58.68421 64.66099 65.69517 LY-163,502•2HCl 73.37074 60.97297 59.42857 66.48606 Dipropyldopamine•HBr 69.17914 74.36364 65.5 51.82253 B-HT 920•2HCl 75.67606 46.90411 64.78571 76.23529 Piribedil•2HCl 61.58491 85.76119 59.05263 59.63793 (+)-UH 232 maleate 61.9521 68 71.84615 67.26608 Pergolide mesylate 82.48669 69.37255 70.66667 65.57576 (−)-Quinpirole•HCl 80.11538 73.14851 80.53731 68.54475 R(−)-2,11-dihydroxy-10- 85.18283 74.66667 80.72727 80.45283 methoxyapomorphine•HCl

The ability of selected dopamine receptor agonists to inhibit expression of αSMA is shown in Table 4.

TABLE 4 αSMA intensity (% of DMSO control) Compound IPF-1 IPF-2 IPF-3 IPF-4 DMSO 100 100 100 100 Dihydrexidine•HCl 4.984838 0.04693 0.513048 12.03436 A-77636•HCl•H₂O 5.808214 0.71885 0.067655 12.42941 R(−)-2,10,11-Trihydroxyaporphine•HBr 2.887936 1.698043 7.646467 15.9942 (±)-SKF-82958•HBr 11.77987 4.966244 2.427766 9.32639 A 68930•HCl 12.40274 8.110719 3.24542 7.350053 R(−)-Propylnorapomorphine•HCl 5.081187 1.878811 5.560538 19.95755 CY 208-243 21.01121 2.279688 2.610795 9.642782 R(+)-6-bromo-APB•HBr 11.25082 2.507086 0.850571 22.81622 R(−)-2,10,11-Trihydroxy-N-propyl- 4.894616 8.436081 2.988195 25.60956 noraporphine•HBr (R)-(−)-Apomorphine•HCl 7.067375 1.172584 0.961841 33.67351 6,7-ADTN•HBr 37.14071 4.182238 9.908606 4.887935 Dopamine•HCl 35.24978 8.085496 26.56503 5.368141 N-Methyldopamine•HCl 43.19963 17.66949 28.99207 14.65662 SKF 38393•HBr 37.06277 26.81876 31.24847 20.7973 Mesulergine•HCl 35.50719 23.27498 40.01858 19.75155 Dipropyldopamine•HBr 48.72246 28.11431 40.33584 25.85713 Bromocriptine mesylate 54.0785 53.62163 61.14226 34.0979 Pergolide mesylate 65.01308 51.5948 63.61159 48.1321 (±)-2-(N-phenylethyl-N-propyl)amino-5- 65.43466 52.77428 60.4844 51.10147 hydroxytetralin•HCl Piribedil•2HCl 70.43668 61.52793 71.47113 41.19743 Cabergoline 76.91545 48.07228 77.14592 45.8402 Fenoldopam•HCl 77.33538 45.27138 69.40569 63.35173 B-HT 920•2HCl 74.97842 58.97091 70.11813 54.23016 Ropinirole HCl 72.18286 53.45179 78.99604 61.9799 PD 168077 maleate 75.26704 56.14917 75.76791 66.49739 (+)-UH 232 maleate 82.91486 63.85248 85.81355 65.71164 LY-163,502•2HCl 88.98983 72.85814 75.30018 86.7894 (−)-Quinpirole•HCl 84.60746 67.1295 85.40936 95.1307 Pramipexole Dihydrochloride Monohydrate 87.61601 73.21711 82.7588 89.93775 (±)-PD 128,907•HCl 83.67743 85.45062 94.2538 75.15364 R(−)-2,11-dihydroxy-10- 91.84016 92.79975 81.85245 85.60691 methoxyapomorphine•HCl 3-Hydroxyphenethylamine•HCl 88.58294 96.744 82.62473 99.8315 4-Hydroxyphenethylamine•HCl 99.7156 93.43022 95.69317 88.2748

Example 3—Dopamine Receptor Agonist (e.g., DHX) Reduces Fibroblast Activation and Matrix Deposition

To test whether DHX-mediated inhibition of YAP/TAZ nuclear localization translates into altered mesenchymal cell activation, we first demonstrated that expression of hallmark profibrotic genes CTGF, COL1A1, ACTA2, and FN1 was reduced in IPF patient-derived lung fibroblasts by DHX treatment, recapitulating effects of YAP/TAZ knockdown (See e.g., Ref. 1-4) (FIG. 11). These effects could be blocked with a DRD1 antagonist (FIG. 11) as well as DRD1-siRNA (FIG. 27-28). Stimulating fibroblasts cultured on stiff tissue culture plastic for 72 hours with TGFβ further enhances their myofibroblastic transition, as detected by αSMA+ stress fibers; treating fibroblasts with DHX from 48-72 hours in the presence of TGFβ reversed this transition (FIG. 12). Similarly, DHX dose-dependently reversed fibroblast-mediated, TGFβ-stimulated accumulation of collagen I and fibronectin (FIG. 13). To validate that this effect is dependent on inhibition of YAP/TAZ, NIH-3T3 cells were employed which stably express a doxycycline-inducible, constitutively active, mutant TAZ (TAZ4SA) (See, e.g., Ref. 2, 36). In these cells, DHX had no effect on profibrotic gene expression or extracellular matrix accumulation (FIG. 29, 30). Finally, traction force microscopy (TFM) demonstrated that DHX significantly and dose-dependently reduced the contractile forces generated by fibroblasts (FIG. 14).

Example 4—Extracellular Matrix Remodeling by Dopamine Receptor Agonist (e.g., DHX)

The results above were consistent with DHX not only attenuating key aspects of fibroblast pro-fibrotic activation, but potentially shifting their phenotype toward one that promotes fibrosis clearance and resolution. To test this concept directly, an in vitro matrix remodeling assay was developed. Fibroblasts (NIH-3T3 cells) were first plated at confluence (A and B in FIG. 15), following an approach developed for studying cell-derived matrices. Cells on both plates were cultured with TGFβ and ascorbic acid to promote matrix synthesis and deposition. After 72 hours the stiffness of the cells and their cell-derived matrix were measured using atomic force microscopy (AFM). To test the ability of DHX to induce cell-mediated matrix remodeling, TGFβ and ascorbic acid were maintained but also DHX was added to B (vehicle control was added to A) for an additional 72 hours before probing the matrix again with AFM. While the cells and matrix in control plate A continued to stiffen over time, DHX treatment effectively reversed this trend and significantly reduced the observed stiffness. To confirm that DHX introduced a matrix remodeling effect, the matrices were decellularized and plated low passage primary human adult lung fibroblasts for 24 hours onto the decellularized matrices, then RNA was isolated to measure changes in profibrotic gene expression. CTGF, COL1A1, ACTA2 and FN1 expression were all decreased in the cells plated onto the matrix which had previously been treated with DHX, identifying a pharmaco-footprint left behind in the extracellular matrix by cellular remodeling.

Based on these matrix remodeling effects of DHX, it was determined whether efficacy of this pathway extends to inducing matrix degradation/remodeling action in fibroblasts which could reverse the disease rather than simply slow its progression (an important utility of fibroblasts). First, the effect or dopamine receptor agonist (e.g., DHX) was investigated on expression of genes associated with matrix remodeling (FIG. 3f ). IPF patient derived fibroblasts treated with TGFβ showed enhanced expression of matrix crosslinking genes and inhibitors of matrix protease enzymes but also showed reduced expression of several genes associated with matrix clearance (FIG. 16). In each case, DHX treatment reversed the effects of TGFβ, reducing expression of crosslinking and protease inhibitors but enhancing expression of matrix degradation associated enzymes. There doesn't appear to be a single driver gene by which DHX effects matrix remodeling, suggesting that the observed effect is a broader fibroblast program shift.

Example 5—Dopamine Receptor Agonist (e.g., DHX) Efficacy In Vivo

Bleomycin model of pulmonary fibrosis was used to test the efficacy of DHX in vivo in experimental fibrosis. Mice were administered bleomycin intratracheally at Day 0. On Day 10 injury and inflammation typically subside and fibrosis is ongoing. At Day 10 mice were randomized into two groups, one receiving DHX (5 mg/kg once daily i.n.) and the other, vehicle control. The Bleo DHX group lost significantly less weight than the Bleo control group (FIG. 17). At day 24 the mice were sacrificed and compared using histology, hydroxyproline and qPCR. Histologically, the Bleo DHX group was nearly completely protected from lung remodeling compared to the Bleo control group and sham uninjured mice, as assessed by a blinded pathologist (FIG. 18). Total collagen in the lungs of Bleo DHX mice was nearly identical to sham treated mice, and significantly reduced compared to Bleo control mice (FIG. 19). Bleomycin increased staining for YAP and TAZ in the lungs and this was attenuated by DHX treatment (FIG. 20). The Bleo Control group also showed enhanced transcript levels for profibrotic genes Acta2, Ctgf, Fn1, Col1a1, and Col1a2, as well as Yap and Taz themselves, all of which were significantly attenuated in the Bleo DHX group (FIG. 21). To assess whether DHX adversely effects lung remodeling in the absence of fibrosis, we also exposed control mice to DHX following an identical time course and route of exposure. The lungs of Sham DHX mice did not differ from those of control mice using any of these measurements (FIG. 31-34).

Example 6—Effect of Dopamine Receptor Agonist (e.g., DHX) on Hepatic Stellate Cells

Based on the efficacy of DHX in attenuating YAP/TAZ nuclear localization across an array of mesenchymal cells, we sought to extend our findings to hepatic stellate cells and liver fibrosis. Preferential expression of DRD1 in hepatic stellate cells (HSCs) compared to hepatocytes (FIG. 35-38) was confirmed, with results similar to those obtained for lung tissue in the previous examples. Ability of DHX to reduce TGFβ-mediated HSC expression of SMA and FN protein was then tested by western blotting, and observed significant reversal of both. Efficacy of DHX in the bile duct ligation model of cholestatic injury and liver fibrosis was then tested. Bile duct ligation was performed at Day 0 and treatment with DHX or vehicle began at Day 7 and continued for 14 days. DHX significantly improved histological fibrosis caused by the BDL and exhibited a trend toward reduced collagen deposition (FIG. 35-38).

Previous work demonstrated that TAZ mediates fibrotic effects in hepatocytes in a model of non-alcoholic steatohepatitis (Ref. 37), that verteporfin (an inhibitor of YAP/TAZ-TEAD interactions) has limited beneficial effects in models of liver fibrosis models (Ref. 19), and that YAP/TAZ are essential in liver regeneration (nonspecific YAP knockdown in liver promotes hepatocyte necrosis (Ref. 38)). The experimental results presented here demonstrate the efficacy of a GPCR-based approach to selective inhibition of YAP/TAZ in experimental liver fibrosis.

Example 7—DOPA Decarboxylase is Decreased in IPF, and Correlates with Worsening Disease Severity

It was discovered that IPF patient lungs express less dopa decarboxylase (DDC) (enzyme that takes part in dopamine synthesis) than non-IPF lungs and the lower level of DDC expression correlates with decreased lung function consistent with an endogenous, protective role for dopamine signaling that is lost in pulmonary fibrosis (FIG. 61A-C). In support of this, it was also shown that dopamine is antifibrotic in in vitro assessments of fibroblast activity (FIG. 62A-C).

Taken together, the experimental results and data presented in Examples 1-7 demonstrate that GPCR agonism (e.g., dopamine receptor agonism) can be used to pharmacologically target YAP and TAZ in selective cell populations to exert beneficial effects on tissue fibrosis. Ga_(s) agonism (e.g., dopamine receptor agonism) and YAP/TAZ inhibition reverses the matrix deposition and stiffening phenotype of activated fibroblasts toward a matrix remodeling phenotype that promotes fibrosis resolution, showing that Ga_(s) agonism (e.g., dopamine receptor agonism) and YAP/TAZ inhibition is an valuable approach to treat patients with fibrotic diseases.

Example 8—Compounds CTC-1, CTC-2, CTC-3, CTC-6, and CTC-7 Potently Inhibit Models of Tissue Fibrosis

As shown in FIGS. 66-76, compounds CTC-1, CTC-2, CTC-3, and CTC-6 inhibit YAP/TAZ nuclear localization, inhibit fibroblast proliferation, inhibit fibroblast activation, inhibit Collagen I deposition, and inhibition of profibrotic gene expression.

Example 9—Bioactivity of Exemplified Compounds

The compounds described in this disclosure are useful as D1 dopamine receptor agonists for the treatment of idiopathic pulmonary fibrosis. This receptor is preferentially expressed on lung fibroblasts relative to other major resident cell types, providing a mechanism to selectively inhibit the YAP/TAZ transcription program in lung fibroblasts to promote antifibrotic/pro-resolving phenotypes. As shown in the previous example, compounds CTC-3 and CTC-6 potently inhibit the localization of YAP/TAZ in cultured lung fibroblasts (IC₅₀ 50-100 nM) and their physical and chemical properties suggest marginal ability to cross the blood-brain-barrier. The compounds of this example maintain or improve the potency of CTC-3/6 while enhancing the intrinsic activity (efficacy) at the D1 receptor.

Most exemplified D1 receptor agonists of the present disclosure contain a catechol moiety. At physiological pH, catechols are sometimes rapidly oxidized into quinones, and for several D1 agonists, a bulk of the drug clearance is a result of this oxidation, not liver metabolism. Fenoldopam is a clinically approved dopamine receptor agonist for the treatment of acute hypertension which contains the chloride substituted catechol ring. The chlorine substitution sometimes has a protecting effect which results in fenoldopam being stable at physiological pH and no reported metabolism through oxidation. Of note, chloride substitution at this site also enhances dopamine receptor potency. In sum, halogen substitutions to the catechol moiety in the exemplified compounds enhances their efficacy and plasma stability, e.g., by preventing oxidation. Chemical structures of exemplified compounds are shown in the tables below.

TABLE 9a

TABLE 9b

TABLE 9c

TABLE 9d

(example 2)

TABLE 9e

(example 1)

TABLE 9f

TABLE 9g

TABLE 9h

Example 10—Compound 1 (MS-9) Potently Inhibits Models of Tissue Fibrosis

As shown in FIG. 90, compound 1 (MS-9) potently inhibits fibroblast proliferation. Fibroblasts were stimulated with 2 ng/mL TGFβ and treated with compound 1 (MS-9) at the indicated concentration (0.1 μM, 1 μM, and 10 μM). Proliferation determined by fixing and counting DAPI nuclei using a Cytation.

Alpha-smooth muscle actin (αSMA) staining is a well-defined marker of fibroblast activation observed in wound healing and tissue fibrosis. As shown in FIG. 91A, compound 1 (MS-9) potently inhibits fibroblast activation. Fibroblasts were stimulated with 2 ng/mL TGFβ and treated with the indicated concentration of compound 1 (MS-9) every 48 hours (IC₅₀ is 1.1 μM). Imaging and quantification of αSMA intensity performed through automation using a Cytation 5.

As shown in FIG. 91B, compound 1 (MS-9)) potently inhibits collagen 1 deposition. Fibroblasts were stimulated with 2 ng/mL TGFβ and treated with the indicated concentration of compound 1 every 48 hours (IC₅₀ is 0.7 μM). Imaging and quantification of collagen I intensity performed through automation using a LI-COR Odyssey.

In cell culture studies, MS-9 potently blocks YAP/TAZ nuclear localization in fibroblasts but promotes YAP/TAZ nuclear localization in epithelial cells and effectively inhibits fibroblast activation in models of lung fibrosis. See FIGS. 92A-92C.

At physiological pH apomorphine is oxidized into the inactive quinone, representing a majority of its metabolism:

MS-21-9 has a chloride substitution, which stabilizes the catechol while enhancing dopamine receptor potency. As such, MS-21-9 has a longer half-life compared to compounds lacking halogen on the catechol moiety.

Additional exemplified compounds are shown in the table below:

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Numbered Paragraphs

In some embodiments, the invention of the present disclosure can be described by reference to the following numbered paragraphs:

Paragraph 1. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, and 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S;

wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²; and

each R² is independently selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

Paragraph 2. The compound of paragraph 1, wherein R¹ is 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 3. The compound of paragraph 2, wherein R¹ is selected from pyridinyl, pyrimidinyl, and pyrazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 4. The compound of paragraph 2, wherein R¹ is pyridinyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 5. The compound of paragraph 4, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

Paragraph 6. The compound of paragraph 1, wherein R¹ is 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 7. The compound of paragraph 6, wherein R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, and pyrrolidinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 8. The compound of paragraph 6, wherein R¹ is tetrahydropyranyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².

Paragraph 9. The compound of paragraph 8, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

Paragraph 10. The compound of paragraph 1, wherein R¹ is selected from HO—C₁₋₆ alkyl and NH₂—C₁₋₆ alkyl.

Paragraph 11. The compound of paragraph 1, wherein R¹ is HO—C₁₋₆ alkyl.

Paragraph 12. The compound of any one of paragraphs 1-9, wherein each R² is independently selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

Paragraph 13. The compound of paragraph 1, wherein the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

Paragraph 14. A pharmaceutical composition comprising a compound of any one of paragraphs 1-13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Paragraph 15. A method of:

-   -   agonizing a Gα_(S) protein coupled receptor in a cell; and/or     -   promoting YAP/TAZ phosphorylation in a cell; and/or     -   inhibiting YAP/TAZ function in a cell; and/or     -   inhibiting expression of a profibrotic gene in a cell; and/or     -   reducing nuclear localization of YAP/TAZ in a cell; and/or     -   inhibiting expressing of α-smooth muscle actin (αSMA) in a cell;         and/or     -   inhibiting extra-cellular matrix production and deposition by a         cell; and/or     -   enhancing extra-cellular matrix degradation by a cell;

the method comprising contacting the cell with an effective amount of a compound of any one of paragraphs 1-13, or a pharmaceutically acceptable salt thereof.

Paragraph 16. The method of paragraph 15, wherein the profibrotic gene is selected from the group consisting of: CTGF, COL1A1, ACTA2, and FN.

Paragraph 17. The method of paragraph 15 or 16, wherein the Gα_(S) protein coupled receptor is a dopamine receptor.

Paragraph 18. The method of paragraph 17, wherein the dopamine receptor is dopamine receptor D1 (DRD1).

Paragraph 19. The method of paragraph 18, wherein the method comprises selectively agonizing D1 dopamine receptor, as compared to D2, D3, D4, or D5 dopamine receptor, or any combination thereof.

Paragraph 20. The method of any one of paragraphs 15-19, wherein the cell is a mesenchymal cell.

Paragraph 21. The method of paragraph 20, wherein the mesenchymal cell is selected from a fibroblast and a stellate cell.

Paragraph 22. A method of treating or preventing a fibrotic pathology, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of paragraphs 1-13, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of paragraph 14.

Paragraph 23. The method of paragraph 22, wherein the fibrotic pathology is interstitial lung disease (ILD).

Paragraph 24. The method of paragraph 22, wherein the fibrotic pathology is selected from pulmonary fibrosis (PF) and idiopathic pulmonary fibrosis (IPF).

Paragraph 25. The method of paragraph 22, wherein the fibrotic pathology is selected from liver tissue fibrosis, cardiac fibrosis, kidney fibrosis, and skin tissue fibrosis.

Paragraph 26. The method of any one of paragraphs 22-25, further comprising administering to the subject a therapeutically effective amount of an additional therapeutic agent useful in treating a fibrotic pathology.

Paragraph 27. The method of paragraph 26, wherein the additional therapeutic agent is dopamine, or a pharmaceutically acceptable salt thereof.

Paragraph 28. The method of paragraph 26, wherein the additional therapeutic agent is a dopamine receptor agonist.

Paragraph 29. The method of claim 28, wherein the dopamine receptor agonist is selected from: ABT-413, A-86929, dihydrexidine (DHX), dinapsoline, dinoxyline, doxanthrine, SKF-81297, SKF-82958, SKF-38393, fenoldopam, 6-Br-APB, stepholidine, A-68930, A-77636, CY-208-243, SKF-89145, SKF-89626, 7,8-dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline, cabergoline, pergolide, R(−)-2,10,11-trihydroxyaporphine, (R)-(−)-apomorphine, R(−)-propylnorapomorphine, R(+)-6-bromo-APB, R(−)-2,10,11-trihydroxy-N-propyl-noraporphine, 6,7-ADTN, mesulergine, N-methyldopamine, 4-hydroxyphenethylamine, cabergoline, 3-hydroxyphenethylamine, pramipexole, PD-168077, fenoldopam, (+)-PD 128-907, (+)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin, bromocriptine, ropinirole, LY-163-502, dipropyldopamine, B-HT 920, piribedil, (+)-UH 232, pergolide, (−)-quinpirole, R(−)-2,11-dihydroxy-10-methoxyapomorphine, or a pharmaceutically acceptable salt thereof.

Paragraph 30. A compound of Formula (II):

-   -   or a pharmaceutically acceptable salt thereof, wherein:     -   R¹ is selected from H and C₁₋₃ alkyl, wherein said C₁₋₃ alkyl is         optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, or         di(C₁₋₃ alkyl)amino;     -   R², R³, and R⁴ are each independently selected from H, OH, SH,         NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃         haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted         with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino;     -   provided that:         -   (i) at least one of R², R³, and R⁴ is not H;         -   (ii) if R² is H and R³ is OH, then R⁴ is not H or OH; and         -   (iii) if R² is OH, then at least one of R³ and R⁴ is not H.

Paragraph 31. The compound of paragraph 30, wherein R¹ is H.

Paragraph 32. The compound of paragraph 30, wherein R¹ is C₁₋₃ alkyl.

Paragraph 33. The compound of paragraph 30, wherein R¹ is selected from HO—C₁₋₃ alkyl and NH₂—C₁₋₃ alkyl.

Paragraph 34. The compound of any one of paragraphs 30-33, wherein at least one of R², R³, and R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

Paragraph 35. The compound of any one of paragraphs 30-33, wherein at least one of R², R³, and R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

Paragraph 36. The compound of any one of paragraphs 30-33, wherein at least one of R², R³, and R⁴ is C₁₋₃ alkyl.

Paragraph 37. The compound of any one of paragraphs 30-33, wherein:

-   -   R³ is OH; and     -   R² is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃         alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃         alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃         alkylamino and di(C₁₋₃ alkyl)amino.

Paragraph 38. The compound of any one of paragraphs 30-33, wherein:

-   -   R³ is OH; and     -   R² is selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and         NH₂—C₁₋₃ alkyl.

Paragraph 39. The compound of any one of paragraphs 30-33, wherein:

-   -   R³ is OH; and     -   R⁴ is selected from SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃         alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃         alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃         alkylamino and di(C₁₋₃ alkyl)amino.

Paragraph 40. The compound of any one of paragraphs 30-33, wherein:

-   -   R³ is OH; and     -   R⁴ is selected from NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃         alkyl.

Paragraph 41. The compound of any one of paragraphs 30-33, wherein:

-   -   R⁴ is OH; and     -   R³ is selected from H, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃         alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃         alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃         alkylamino and di(C₁₋₃ alkyl)amino.

Paragraph 42. The compound of any one of paragraphs 30-33, wherein:

-   -   R⁴ is OH; and     -   R³ is selected from H, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and         NH₂—C₁₋₃ alkyl.

Paragraph 43. The compound of any one of paragraphs 30-33, wherein:

-   -   R² is OH; and     -   at least one of R³ and R⁴ is selected from OH, SH, NH₂, C₁₋₃         alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl,         wherein said C₁₋₃ alkyl is optionally substituted with OH, SH,         NH₂, C₁₋₃ alkylamino and di(C₁₋₃ alkyl)amino.

Paragraph 44. The compound of any one of paragraphs 30-33, wherein:

-   -   R² is OH; and     -   at least one of R³ and R⁴ is selected from OH, NH₂, C₁₋₃ alkyl,         HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.

Paragraph 45. The compound of paragraph 30, wherein the compound of Formula (II) is:

or a pharmaceutically acceptable salt thereof.

Paragraph 46. The compound of paragraph 30, wherein the compound of Formula (II) is:

or a pharmaceutically acceptable salt thereof.

Paragraph 47. A pharmaceutical composition comprising a compound of any one of paragraphs 30-46, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Paragraph 48. A method of

-   -   agonizing a Gα_(S) protein coupled receptor in a cell; and/or     -   promoting YAP/TAZ phosphorylation in a cell; and/or     -   inhibiting YAP/TAZ function in a cell; and/or     -   inhibiting expression of a profibrotic gene in a cell; and/or     -   reducing nuclear localization of YAP/TAZ in a cell; and/or     -   inhibiting expressing of α-smooth muscle actin (αSMA) in a cell;         and/or     -   inhibiting extra-cellular matrix production and deposition by a         cell; and/or     -   enhancing extra-cellular matrix degradation by a cell;

the method comprising contacting the cell with an effective amount of a compound of any one of paragraphs 30-46, or a pharmaceutically acceptable salt thereof.

Paragraph 49. The method of paragraph 48, wherein the profibrotic gene is selected from the group consisting of: CTGF, COL1A1, ACTA2, and FN.

Paragraph 50. The method of paragraph 48 or 49, wherein the Gα_(S) protein coupled receptor is a dopamine receptor.

Paragraph 51. The method of paragraph 50, wherein the dopamine receptor is dopamine receptor D1 (DRD1).

Paragraph 52. The method of paragraph 51, wherein the method comprises selectively agonizing D1 dopamine receptor, as compared to D2, D3, D4, or D5 dopamine receptor, or any combination thereof.

Paragraph 53. The method of any one of paragraphs 48-52, wherein the cell is a mesenchymal cell.

Paragraph 54. The method of paragraph 53, wherein the mesenchymal cell is selected from a fibroblast and a stellate cell.

Paragraph 55. A method of treating or preventing a fibrotic pathology, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of paragraphs 30-46, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of paragraph 47.

Paragraph 56. The method of paragraph 55, wherein the fibrotic pathology is interstitial lung disease (ILD).

Paragraph 57. The method of paragraph 56, wherein the fibrotic pathology is selected from pulmonary fibrosis (PF) and idiopathic pulmonary fibrosis (IPF).

Paragraph 58. The method of paragraph 55, wherein the fibrotic pathology is selected from liver tissue fibrosis, cardiac fibrosis, kidney fibrosis, and skin tissue fibrosis.

Paragraph 59. The method of any one of paragraphs 55-58, further comprising administering to the subject a therapeutically effective amount of an additional therapeutic agent useful in treating a fibrotic pathology.

Paragraph 60. The method of paragraph 59, wherein the additional therapeutic agent is dopamine, or a pharmaceutically acceptable salt thereof.

Paragraph 61. The method of paragraph 59, wherein the additional therapeutic agent is a dopamine receptor agonist.

Paragraph 62. The method of paragraph 61, wherein the dopamine receptor agonist is selected from: ABT-413, A-86929, dihydrexidine (DHX), dinapsoline, dinoxyline, doxanthrine, SKF-81297, SKF-82958, SKF-38393, fenoldopam, 6-Br-APB, stepholidine, A-68930, A-77636, CY-208-243, SKF-89145, SKF-89626, 7,8-dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline, cabergoline, pergolide, R(−)-2,10,11-trihydroxyaporphine, (R)-(−)-apomorphine, R(−)-propylnorapomorphine, R(+)-6-bromo-APB, R(−)-2,10,11-trihydroxy-N-propyl-noraporphine, 6,7-ADTN, mesulergine, N-methyldopamine, 4-hydroxyphenethylamine, cabergoline, 3-hydroxyphenethylamine, pramipexole, PD-168077, fenoldopam, (±)-PD 128-907, (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin, bromocriptine, ropinirole, LY-163-502, dipropyldopamine, B-HT 920, piribedil, (+)-UH 232, pergolide, (−)-quinpirole, R(−)-2,11-dihydroxy-10-methoxyapomorphine, or a pharmaceutically acceptable salt thereof.

Other Embodiments

It is to be understood that while the present application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present application, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 to 5 heteroatoms selected from N, O, and S, and 3-10-membered heterocycloalkyl ring comprising 1 to 3 heteroatoms independently selected from N, O, and S; wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²; each R² is independently selected from halo, OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, C₁₋₃ alkoxy, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino; and R³ is selected from H and halo.
 2. The compound of claim 1, wherein the compound of Formula (I) has formula

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selected from HO—C₁₋₆ alkyl, NH₂—C₁₋₆ alkyl, 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, and 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S; wherein said heteroaryl ring and heterocycloalkyl ring are each optionally substituted with 1, 2, or 3 substituents independently selected from R²; and each R² is independently selected from OH, SH, NH₂, C₁₋₃ alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkyl, and C₁₋₃ haloalkyl, wherein said C₁₋₃ alkyl is optionally substituted with OH, SH, NH₂, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.
 3. (canceled)
 4. The compound of claim 1, wherein R¹ is 5-6-membered heteroaryl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².
 5. The compound of claim 1, wherein R¹ is selected from pyridinyl, pyrimidinyl, pyrazinyl, diazinyl, triazinyl, tetrazinyl, and pentazinyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².
 6. (canceled)
 7. The compound of claim 1, wherein R¹ is pyridinyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².
 8. The compound of claim 7, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim 7, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 10. (canceled)
 11. The compound of claim 1, wherein R¹ is 3-7-membered heterocycloalkyl ring comprising 1 or 2 heteroatoms selected from N, O, and S, which is optionally substituted with 1, 2, or 3 substituents independently selected from R².
 12. The compound of claim 11, wherein R¹ is selected from tetrahydropyranyl, piperidinyl, tetrahydrofuranyl, pyrrolidinyl, pyranyl, morpholinyl, oxazinyl, dioxanyl, dioxinyl, diazinanyl, triazinanyl, trioxanyl, azepanyl, azepinyl, oxepanyl, oxepinyl, diazepanyl, diazepinyl, azocanyl, azocinyl, oxocanyl, oxocinyl, azonanyl, azoninyl, oxonanyl, and oxoninyl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from R².
 13. (canceled)
 14. The compound of claim 11, wherein R¹ is tetrahydropyranyl, optionally substituted with 1, 2, or 3 substituents independently selected from R².
 15. The compound of claim 14, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 16. The compound of claim 14, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 17. The compound of claim 12, wherein the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.
 18. The compound of claim 1, wherein R¹ is selected from HO—C₁₋₆ alkyl and NH₂—C₁₋₆ alkyl. 19-20. (canceled)
 21. The compound of claim 1, wherein each R² is independently selected from OH, NH₂, C₁₋₃ alkyl, HO—C₁₋₃ alkyl, and NH₂—C₁₋₃ alkyl.
 22. (canceled)
 23. The compound of claim 1, wherein R³ is selected from H, Cl, F, and Br.
 24. The compound of claim 1, wherein the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.
 25. The compound of claim 1, wherein the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.
 26. The compound of claim 1, wherein the compound of Formula (I) is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof. 27-105. (canceled)
 106. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 